Cns targeting aav vectors and methods of use thereof

ABSTRACT

The invention in some aspects relates to recombinant adeno-associated viruses useful for targeting transgenes to CNS tissue, and compositions comprising the same, and methods of use thereof. In some aspects, the invention provides methods and compositions for treating CNS-related disorders.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of U.S.application Ser. Nos. 16/234,819 and 16/234,834, filed Dec. 28, 2018,which are each a continuation under 35 U.S.C. § 120 of U.S. applicationSer. No. 15/613,646, filed Jun. 5, 2017, which is a continuation of U.S.application Ser. No. 14/445,670, filed Jul. 29, 2014, which is acontinuation of U.S. application Ser. No. 13/642,719, filed Jan. 7,2013, which is a national stage filing under 35 U.S.C. § 371 ofInternational Patent Application Serial No. PCT/US2011/033616, filedApr. 22, 2011, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Application Ser. No. 61/327,627, filed Apr. 23, 2010. The entire contentof each of the above-referenced applications is incorporated herein byreference.

FIELD OF THE INVENTION

The invention in some aspects relates to recombinant adeno-associatedviruses useful for targeting transgenes to CNS tissue, and compositionscomprising the same, and methods of use thereof.

BACKGROUND OF THE INVENTION

Gene therapy has been investigated for delivery of therapeutic genes tothe CNS cells for treatment of various CNS disease, e.g., Canavandisease, ALS, Parkinson disease (PD), etc. In some limited cases,therapeutic benefits have been observed using certain viruses, e.g.,recombinant adenovirus (rAd), lentivirus (LV) and adeno-associated virus(AAV) to express a variety of therapeutic genes. AAV2 has been used inclinical trials for treatment of PD and Leber congenital amaurosis (aneye disease) and preliminary findings suggest symptomatic improvementswithout noticeable toxicity [2-4].

However, AAV-based gene therapy to treat CNS disease has still facedmajor obstacle. Many CNS diseases including, for example, ALS affectboth cortical and spinal motor neurons that are distributed in a verybroad area in the CNS. It has frequently been the case that viralvectors injected into CNS tissue transduce cells only in the vicinity ofthe injection site, have a very limited spread and generally have notimpacted the lifespan in CNS disease animal models [See, e.g., Ref. 5].Still, a variety of other viral administration methods have been tested.One example, involves injecting the viral particles into skeletal muscleand allowing the nerve terminals to internalize the viral genome, whichis then retrogradely transported back to the spinal motor neurons. Thisapproach has shown some positive results in certain mouse models [68].However, to apply this method in larger mammals, like adult humans,would be impractical. Overall, the transduction efficiency observed withmuscle injection is relatively low. Some investigators have tried toimprove this efficiency by modifying viral capsid proteins with thenerve binding domains of tetanus toxin or botulinum toxin. These effortshave not been fruitful due to various technical difficulties. Anotherproblem with muscle injection in larger mammals, is a need for largedoses, which is technically challenging, expensive, and carries a highrisk for adverse effects, ranging from immune reaction to transductionof unintended cells (e.g., germ cells).

Another method that has been evaluated for delivering transgenes intomotor neurons is to inject the virus into large nerves, which maximizesthat exposure of the virus to motor axons, allowing the motor neurons tointernalize the viral genome and retrogradely transport them back to thecell body. This method has been demonstrated to be more efficient intransducing motor neurons than muscle injection [9]. Still, to implementa method such as this in larger mammals would be challenging.

SUMMARY OF THE INVENTION

Aspects of the invention, are based on the discovery of recombinant AAVsthat achieve wide-spread distribution throughout CNS tissue of asubject. In some embodiments, the rAAVs spread throughout CNS tissuefollowing direct administration into the cerebrospinal fluid (CSF),e.g., via intrathecal and/or intracerebral injection. In otherembodiments, the rAAVs cross the blood-brain-barrier and achievewide-spread distribution throughout CNS tissue of a subject followingintravenous administration. In some aspects the invention relates torAAVs having distinct central nervous system tissue targetingcapabilities (e.g., CNS tissue tropisms), which achieve stable andnontoxic gene transfer at high efficiencies. Methods involvingco-administration via intrathecal and intracerebral (e.g.,intraventricular) injection of rAAVs are provided in some aspects. Forexample, it has been discovered that rAAVs having a capsid proteincomprising a sequence as set forth in SEQ ID NO: 9 achieves wide-spreaddistribution following intrathecal injection throughout the CNS, andthus, are particularly useful for treating CNS-associated disorders suchas, for example, ALS. In still further aspects of the invention methodsare provided for treating Canavan disease.

According to some aspects of the invention, methods for delivering atransgene to CNS tissue in a subject are provided. In some embodiments,the methods comprise administering an effective amount of a rAAV byintrathecal administration, wherein the rAAV comprises (i) a capsidprotein comprising a sequence as set forth in SEQ ID NO: 9 and (ii) anucleic acid comprising a promoter operably linked with a transgene. Insome embodiments, the methods further comprise administering aneffective amount of the rAAV by intracerebral administration. In someembodiments, the methods comprise administering an effective amount of arAAV by intrathecal administration and by intracerebral administration,wherein the rAAV infects cells of CNS tissue in the subject andcomprises a nucleic acid comprising a promoter operably linked with atransgene. In certain embodiments, the intracerebral administration isan intraventricular administration. In one embodiment, theintraventricular administration is an administration into a ventricularregion of the forebrain of the subject. In certain embodiments, theintrathecal administration is in the lumbar region of the subject. Insome embodiments, the dose of the rAAV for intrathecal administration isin a range of 10¹⁰ genome copies/subject to 10¹¹ genome copies/subject.In some embodiments, the dose of the rAAV for intrathecal administrationis in a range of 10¹¹ genome copies/subject to 10¹² genomecopies/subject. In some embodiments, the dose of the rAAV forintrathecal administration is in a range of 10¹² genome copies/subjectto 10¹³ genome copies/subject. In some embodiments, the dose of the rAAVfor intrathecal administration is in a range of 10¹³ genomecopies/subject to 10¹⁴ genome copies/subject. In some embodiments, thedose of the rAAV for intracerebral administration is in a range of 10¹⁰genome copies/subject to 10¹¹ genome copies/subject. In someembodiments, the dose of the rAAV for intracerebral administration is ina range of 10¹¹ genome copies/subject to 10¹² genome copies/subject. Insome embodiments, the dose of the rAAV for intracerebral administrationis in a range of 10¹² genome copies/subject to 10¹³ genomecopies/subject. In some embodiments, the dose of the rAAV forintracerebral administration is in a range of 10¹³ genome copies/subjectto 10¹⁴ genome copies/subject. In some embodiments, the dose of the rAAVfor intracerebral or intrathecal administration is formulated forinjection of a volume in a range of 1 μl to 10 μl. In some embodiments,the dose of the rAAV for intracerebral or intrathecal administration isformulated for injection of a volume in a range of 10 μl to 100 μl. Insome embodiments, the rAAV for the intracerebral or intrathecaladministration is formulated for injection of a volume in a range of 100μl to 1 ml. In some embodiments, the rAAV for the intracerebral orintrathecal administration is formulated for injection of a volume of 1ml or more. In some embodiments, the transgene encodes a reporterprotein. In certain embodiments, the reporter protein is a fluorescentprotein, an enzyme that catalyzes a reaction yielding a detectableproduct, or a cell surface antigen. In certain embodiments, the enzymeis a luciferase, a beta-glucuronidase, a chloramphenicolacetyltransferase, an aminoglycoside phosphotransferase, anaminocyclitol phosphotransferase, or a Puromycin N-acetyl-tranferase. Insome embodiments, the transgene is a CNS-associated gene. In someembodiments, the CNS-associated gene is neuronal apoptosis inhibitoryprotein (NAIP), nerve growth factor (NGF), glial-derived growth factor(GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor(CNTF), tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH), amino aciddecorboxylase (AADC) or aspartoacylase (ASPA). In some embodiments, thetransgene encodes an inhibitory RNA that binds specifically to SOD1 mRNAand inhibits expression of SOD1 in the subject. In some embodiments, theinhibitory RNA is an antisense RNA, a shRNA or a miRNA. In someembodiments, the inhibitory RNA has a sequence as set forth in SEQ IDNO: 26. Thus, according to some aspects of the invention a nucleic acidcomprising a sequence as set forth in SEQ ID NO: 26 is provided. In someembodiments, a nucleic acid comprising a promoter operably linked with aregion having a sequence as set forth in SEQ ID NO: 26 is provided.

In further aspects of the invention a recombinant AAV comprising anucleic acid comprising a sequence as set forth in SEQ ID NO: 26 isprovided. In some aspects of the invention a recombinant AAV comprisinga nucleic acid comprising a promoter operably linked with a regionhaving a sequence as set forth in SEQ ID NO: 26 is provided. In someembodiments the recombinant AAV further comprises a capsid proteincomprising a sequence as set forth in SEQ ID NO: 9.

According to some aspects of the invention, methods for treatingamyotrophic lateral sclerosis (ALS) in a subject in need thereof areprovided. In some embodiments, the methods comprise administering aneffective amount of a rAAV to CNS tissue of the subject, wherein therAAV comprises (i) a capsid protein comprising a sequence as set forthin SEQ ID NO: 9 and (ii) a nucleic acid comprising a promoter operablylinked with a region encoding an inhibitory RNA that binds specificallyto SOD1 mRNA and inhibits expression of SOD1 in the subject. In someembodiments, the inhibitory RNA is an antisense RNA, a shRNA or a miRNA.In some embodiments, the inhibitory RNA has a sequence as set forth inSEQ ID NO: 26. In some embodiments, the methods comprise administeringan effective amount of a rAAV to the subject, wherein the rAAV comprisesa nucleic acid comprising a promoter operably linked with a regionencoding a sequence as set forth in SEQ ID NO: 26 and wherein the rAAVinfects cells of CNS tissue in the subject.

According to some aspects of the invention, methods for delivering atransgene to a CNS tissue in a subject are provided that compriseadministering an effective amount of a rAAV by intravenousadministration, wherein the rAAV infects cells of CNS tissue in thesubject and comprises a nucleic acid comprising a promoter operablylinked with a transgene. In some embodiments, the dose of the rAAV forintravenous administration is in a range of 10¹⁰ genome copies/subjectto 10¹¹ genome copies/subject. In some embodiments, the dose of the rAAVfor intravenous administration is in a range of 10¹¹ genomecopies/subject to 10¹² genome copies/subject. In some embodiments, thedose of the rAAV for intravenous administration is in a range of 10¹²genome copies/subject to 10¹³ genome copies/subject. In someembodiments, the dose of the rAAV for intravenous administration is in arange of 10¹³ genome copies/subject to 10¹⁴ genome copies/subject. Insome embodiments, the dose of the rAAV for intravenous administration isin a range of 10¹⁴ genome copies/subject to 10¹⁵ genome copies/subject.In some embodiments, the dose of the rAAV for intravenous administrationis in a range of 10¹⁰ genome copies/kg to 10¹¹ genome copies/kg. In someembodiments, the dose of the rAAV for intravenous administration is in arange of 10¹¹ genome copies/kg to 10¹² genome copies/kg. In someembodiments, the dose of the rAAV for intravenous administration is in arange of 10¹² copies/kg to 10¹³ genome copies/kg. In some embodiments,the dose of the rAAV for intravenous administration is in a range of10¹³ genome copies/kg to 10¹⁴ genome copies/kg.

According to some aspects of the invention, methods for delivering atransgene to a CNS tissue in a subject are provided that compriseadministering to the subject an effective amount of a rAAV thatcomprises (i) a capsid protein having a sequence as set forth in any oneof SEQ ID NO: 10 to 12 and (ii) a nucleic acid comprising a promoteroperably linked with a transgene. In some embodiments, the methodscomprise administering to the subject an effective amount of a rAAVcomprising a transgene to a subject, wherein the rAAV comprises a capsidprotein of a AAV serotype, or serotype variant, selected from the groupconsisting of: AAV1, AAV2, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, rh.10,rh.39, rh.43 and CSp3, and wherein: (a) if the AAV serotype is AAV1, theadministration route is not intracerebral, intramuscular, intranerve, orintraventricular and/or the subject is not a mouse, rat or feline; (b)if the AAV serotype is AAV2, the administration route is notintracerebral or intraventricular administration and/or the subject isnot a rat, mouse, feline, marmoset, or macaque; (c) if the AAV serotypeis AAV5, the administration route is not intracerebral orintraventricular administration and/or the subject is not a rat, mouse,or marmoset; (d) if the AAV serotype is AAV6, the subject is not amouse; (e) if the AAV serotype is AAV7, the administration route is notintracerebral administration and/or the subject is not a mouse ormacaque; (f) if the AAV serotype is AAV8, the administration route isnot intracerebral, intraperitoneal, or intravascular administrationand/or the subject is not a mouse or macaque; (g) if the AAV serotype isAAV9, the administration route is not intracerebral or intravascularadministration and/or the subject is not a rat or mouse; and (h) if theAAV serotype is AAVrh.10, the administration route is not intracerebralor intravascular administration and/or the subject is not a rat ormouse. In some embodiments, the AAV serotype, or serotype variant, isselected from AAV1, AAV6, AAV7, rh.39, rh.43, and CSp3, and theadministration route is intravascular administration. In someembodiments, the AAV serotype is AAV7 and the administration route isintravascular administration. In some embodiments, the CNS tissue isselected from cortex, hippocampus, thalamus, hypothalamus, cerebellum,brain stem, cervical spinal cord, thoracic spinal cord, and lumbarspinal cord. In some embodiments, the transgene encodes a reporterprotein. In certain embodiments, the reporter protein is a fluorescentprotein, an enzyme that catalyzes a reaction yielding a detectableproduct, or a cell surface antigen. In certain embodiments, the enzymeis a luciferase, a beta-glucuronidase, a chloramphenicolacetyltransferase, an aminoglycoside phosphotransferase, anaminocyclitol phosphotransferase, or a Puromycin N-acetyl-tranferase. Insome embodiments, the transgene is a CNS-associated gene. In certainembodiments, the CNS-associated gene is neuronal apoptosis inhibitoryprotein (NAIP), nerve growth factor (NGF), glial-derived growth factor(GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor(CNTF), tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH), amino aciddecorboxylase (AADC) or aspartoacylase (ASPA). In some embodiments, therAAV is administered by intravenous injection.

According to some aspects of the invention a rAAV that comprises (i) acapsid protein having a sequence as set forth in any one of SEQ ID NO:10 to 12 and (ii) a nucleic acid comprising a promoter operably linkedwith a CNS-associated gene is provided. In certain embodiments, theCNS-associated gene is neuronal apoptosis inhibitory protein (NAIP),nerve growth factor (NGF), glial-derived growth factor (GDNF),brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF),tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH), amino aciddecorboxylase (AADC) or aspartoacylase (ASPA). In some embodiments, mRNAexpressed from the CNS-associated gene comprises a miRNA binding site ofa miRNA that is preferentially expressed in non-CNS tissue. In certainembodiments, the miRNA binding site is a binding site for miR-122. Incertain embodiments, the miRNA binding site is a binding site for miR-1.In some embodiments, mRNA expressed from the CNS-associated gene doesnot comprise a miRNA binding site of a miRNA that is preferentiallyexpressed in CNS tissue. In some embodiments, the promoter is a CNStissue specific promoter. In certain embodiments, the promoter is apromoter of a gene selected from: neuronal nuclei (NeuN), glialfibrillary acidic protein (GFAP), adenomatous polyposis coli (APC), andionized calcium-binding adapter molecule 1 (Iba-1).

According to some aspects of the invention, a composition comprising arAAV that comprises (i) a capsid protein having a sequence as set forthin SEQ ID NO: 10 to 12 and (ii) a nucleic acid comprising a promoteroperably linked with a CNS-associated gene is provided. In certainembodiments the composition further comprises a pharmaceuticallyacceptable carrier. According to some aspects of the invention, a kitcomprising a container housing the composition is provided. In someembodiments, the container is a sealed vial or ampule. In someembodiments, the container is a syringe.

According to some aspects of the invention, an isolated mammalian cellis provided that comprises a nucleic acid encoding a capsid proteinhaving a sequence as set forth in any one of SEQ ID NO: 10 to 12 and arAAV vector comprising a nucleic acid encoding a CNS-disease associatedgene. In some embodiments, the isolated mammalian cell further comprisesan AAV helper function vector. In some embodiments, isolated mammaliancell further comprises an accessory function vector. In certainembodiments, the CNS-associated gene is neuronal apoptosis inhibitoryprotein (NAIP), nerve growth factor (NGF), glial-derived growth factor(GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor(CNTF), tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH), amino aciddecorboxylase (AADC) or aspartoacylase (ASPA).

According to further aspects of the invention, a method for treatingCanavan disease in a subject in need thereof is provided. In someembodiments, the methods comprise administering an effective amount of arAAV to CNS tissue of the subject, wherein the rAAV comprises (i) acapsid protein other than a capsid protein of AAV serotype 2 and (ii) anucleic acid comprising a promoter operably linked with a regionencoding aspartoacylase (ASPA). Any of the rAAV serotypes disclosedherein may be used in the methods for treating Canavan disease. In someembodiments, the rAAV has a capsid protein having an amino acid sequenceas set forth in SEQ ID NO: 8 or 9 or a variant thereof. In someembodiments, administering is performed intrathecally orintracerebrally. In some embodiments, administering is performedintravascularly.

In some embodiments, the methods comprise administering an effectiveamount of a rAAV to CNS tissue of the subject by a route other thanintracerebral administration, wherein the rAAV comprises a nucleic acidcomprising a promoter operably linked with a region encodingaspartoacylase (ASPA). In some embodiments, the methods compriseadministering an effective amount of a rAAV to CNS tissue of thesubject, wherein the rAAV comprises a nucleic acid comprising a promoteroperably linked with a region encoding aspartoacylase (ASPA); andevaluating kidney function in the subject at least once after theadministration. Any suitable method known in the art may be used toevaluate a subject's kidney function. The evaluation may involve, forexample, an examination of blood or urine urea nitrogen levels, anexamination of blood or urine creatinine levels, a creatinine clearancerate examination, a glomerular filtration rate examination, a filtrationfraction examination, a renal plasma flow examination, an ultrasoundexamination, a microscopic examination of a kidney tissue biopsy or anyother suitable kidney function test. It should be appreciated that insome embodiments an improvement in a subject's kidney function followingtreatment with an rAAV-mediated gene therapy is indicative of efficacyof the gene therapy for treating Canavan disease.

In some embodiments, the methods comprise administering an effectiveamount of a rAAV to CNS tissue of the subject, wherein the rAAVcomprises a nucleic acid comprising a promoter operably linked with aregion encoding aspartoacylase (ASPA); and evaluating vision of thesubject at least once after the administration. Any suitable methodknown in the art may be used to evaluate a subject's vision. Theevaluation may involve, for example, an external examination of the eye,a visual acuity examination, an examination of pupil function, a retinalexamination, an ocular motility examination, an intraocular pressuretest, or an ophthalmoscopic examination. The evaluation may involve adetermination regarding a subject's ability to discriminate colors,objects or shapes or the ability of a subject to discern colors, objectsor shapes from a particular distance. It should be appreciated that insome embodiments an improvement in a subject's vision followingtreatment with an rAAV-mediated gene therapy is indicative of efficacyof the gene therapy for treating Canavan disease.

In some embodiments, the nucleic acid expresses an aspartoacylase (ASPA)mRNA comprising one or more miRNA binds sites for one or more miRNAsthat are more abundant in one or more non-CNS tissues in comparison toCNS tissue. Accordingly, in some embodiments, the mRNA is targeted fordegradation by an miRNA in one or more non-CNS tissues. In someembodiments, the one or more non-CNS tissue is not kidney tissue orretinal tissue. In some embodiments, the one or more miRNAs that aremore abundant in non-CNS tissues in comparison to CNS tissue are atleast two-fold, at least three-fold, at least four-fold, at leastfive-fold, or at least ten-fold more abundant. MiRNAs that are moreabundant in non-CNS tissue versus CNS tissue are known in the art. Forexample, one study discloses the expression levels of more thanthree-hundred different human miRNAs in 40 different tissues, includingCNS tissue, kidney tissue. (See Liang Y, et al., Characterization ofmicroRNA expression profiles in normal human tissues. BMC Genomics. 2007Jun. 12; 8:166, the contents of which relating to miRNAs areincorporated herein by reference). Thus, in some embodiments, theskilled artisan could readily select (e.g., based on data such as aredisclosed in Liang et al.) a suitable miRNA that is more abundant innon-CNS tissue and incorporate a binding site for that miRNA into theencoded mRNA.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1B depict quantitative results of EGFP intensities fromfluorescence microscopic images of a panel of CNS tissue sections fromneonatal mice infected with various rAAVs harboring EGFP expressionvectors. Neonatal mice were administered the rAAVs by intravenousadministration (superfacial temporal vein injection).

FIGS. 2A to 2B depict quantitative results of EGFP intensities fromfluorescence microscopic images of a panel of CNS tissue sections fromadult mice infected with various rAAVs harboring EGFP expressionvectors. Adult mice were administered the rAAVs by intravenousadministration (tail vein injection).

FIG. 3 depicts quantitation of EGFP expression in neonatal mice spinalcord (cervical, thoracic and lumber regions) 21 days post IV injection(5 mice per group). Neonatal mice were administered the rAAVs byintravenous administration (superfacial temporal vein injection).

FIG. 4A depicts results showing that direct CSF injection of AAVrh.10harboring a EGFP gene leads to EGFP expression in broad areas of theCNS. Tissue sections, prepared 60 days post virus injection, frombrainstem, cervical spinal cord, thoracic spinal cord and lumbar spinalcord are shown. Gray/black pixels correspond with EGFP expression.

FIG. 4B depicts results showing that direct CSF injection of AAVrh.10harboring a EGFP gene leads to EGFP expression in astrocytes. Gray/blackpixels correspond with EGFP expression.

FIG. 5A depicts a rAAVrh.10 vector that expresses a microRNA targetingSOD1. The construct employs CAG (chicken β-actin promoter with a CMVenhancer) to drive the expression of EGFP and miR-SOD1 that is locatedin an intron in the 3′-UTR. pA stands for poly A signal. ITRs mark theinverted repeats of the AAV.

FIG. 5B depicts results of experiments that test the silencing potencyof 9 different miRNA constructs, miR-SOD1#5 was found to silence SOD1expression most potently.

FIG. 5C depicts results of experiments in which miR-SOD1#5 was packagedinto AAVrh.10 and used to infect HEK293 cells. Total cellular proteinwas extracted 43 hours after the infection and blotted to detect SOD1.Scr stands for scrambled miRNA; Sod stands for miR-SOD1#5; and C standsfor a control that expresses EGFP only.

FIG. 5D depicts a plasmid map of pAAVscCB6 EGFPmir SODS (5243 bp) (SEQID NO: 21).

FIG. 6A depicts results of gene transfer studies in SOD1 (G93A) mutantmice showing that rAAV rh.10-SOD1 miRNA knockdowns levels of mutant SOD1in astrocytes. Staining in motor neurons was also observed.

FIG. 6B depicts results of gene transfer studies in SOD1 (G93A) mutantmice showing that rAAV rh.10-SOD1 shRNA increases live span, comparedwith a rAAV rh.10-scrambled miRNA.

FIG. 7A depicts quantitation of EGFP expression in cervical, thoracic,and lumber spinal cord tissue compared with life spans individual miceinfected with rAAV rh.10-SOD1 miRNA; rAAV rh.10-SOD1 was administereddirectly to the CSF.

FIG. 7B depicts quantitation of EGFP expression in cervical, thoracic,and lumber spinal cord tissue compared with life spans of individualmice infected with rAAV rh.10-scrambled miRNA; rAAV rh.10-scrambledmiRNA was administered directly to the CSF.

FIG. 8 depicts fluorescence microscopy analysis of mice that have beenadministered intrathecal injections of various AAVs. In this experiment,both AAV9 and AAVrh10 transduce cells along the full length of thespinal cord after a single injection into the CSF in lumbar subarachnoidspace.

FIG. 9 depicts the effects of AAV10-miR-SOD1 treatment. AAV10-miR-SOD1treatment slows disease progression as indicated by the slower loss ofbody weight in treated compared with the control G93A mice.

FIG. 10 depicts fluorescence microscopy analysis of mice that have beenadministered intrathecal injections of various AAVs. In this experiment,AAV9 and AAVrh10 can transduce cells in the broad forebrain areas aftera single injection into the CSF in the third ventricle.

FIG. 11 depicts fluorescence microscopy analysis of tissue sections fromAAV9-injected mice. A single injection of AAV9 and AAVrh10 into thethird ventricle can transduce cells in the broad forebrain areas,including cortex, hippocampus, striatum, thalamus, cerebellum and somescattered cells in the spinal cord. The same general pattern is alsoobserved in AAV10-injected mice.

FIGS. 12A to 12C depict an in vitro validation of artificialmiRNA-binding sites for reporter silencing. Plasmids harboring therAAVCBnLacZ genome with or without miR-1 or miR-122-binding sites weretransfected into human hepatoma (HuH7) cells (FIG. 12A) which expressmiR-122 or cotransfected into 293 cells, together with a plasmidexpressing either pri-miR-122 (FIG. 12B) or pri-miR-1 (FIG. 12C) atmolar ratios of 1:3 (low) or 1:10 (high). 0×: no miRNA-binding site; 1×:one miRNA-binding site; 3×: three miRNA-binding sites. The cells werefixed and stained histochemically with X-gal 48 hours after transfectionand blue cells counted. The percentage of nLacZ-positive cells in eachtransfection were compared to transfection of the control plasmid(prAAVCBnLacZ). CB, chicken β-actin; miR, microRNA; nLacZ,β-galactosidase reporter transgene; rAAV, recombinant adeno-associatedviruses.

FIGS. 13A to 13D depict an in vivo evaluation of endogenousmiRNA-mediated transgene silencing in an rAAV9 transduction. (FIGS. 13Ato 13C) Adult male C588L/6 mice were injected intravenously with 5×10¹³genome copies per kg (GC/kg) each of rAAV9CBnLacZ (no binding site),(FIG. 13A) rAAVCB9nLacZmiR-122BS (one miR-122-binding site) andrAAV9C8nlacZ-(miR-122BS)₃ (three miR-122-binding sites), (FIG. 13B)rAAV9CBnLacZ-miR-1 BS (one miR-1 binding site) andrAAV9CBnLacZ-(miR-1BS)₃ (three miR-1-binding sites, (FIG. 13C)rAAV9CBnLacZ-miR-1BS-miR-122BS (1× each binding site) andrAAV9CBnLacZ-(miR-1BS)₃-(miR-122BS)₃ (three miR-1 and threemiR-122-binding sites). The animals were necropsied 4 weeks after vectoradministration, and appropriate tissues were harvested forcryosectioning and X-gal histochemical staining. miR, microRNA; nLacZ,β-galactosidase reporter transgene; rAAV, recombinant adeno-associatedviruses, and (FIG. 13D) quantification of β-galactosidase activities inliver tissue from animals that received rAAVnLacZ vectors with andwithout miRNA-binding sites.

FIGS. 14A to 14E depict an analysis of expression levels of cognatemiRNA, mRNA, and protein of endogenous miRNA target genes in micetransduced with rAAV9CBnLacZ with or without miRNA-binding sites. Totalcellular RNA or protein was prepared from (FIGS. 14A to 14C) liver or(FIG. 14D) heart. (FIG. 14A) Northern blot detection of miRNAs. U6 smallnuclear RNA provided a loading control. (FIG. 14B) Quantitativereverse-transcription PCR measuring cyclin G1 mRNA. The data arepresented as relative cyclin Gl mRNA levels normalized to β-actin.(FIGS. 14C to 14D) Western blot analyses of protein levels of endogenoustargets of miR-122 and miR-1. Total cellular protein prepared from (FIG.14C) liver or (FIG. 14D) heart was analyzed for cyclin G1 andcalmodulin. (FIG. 14E) Serum cholesterol levels. Serum samples from micethat received rAAV9 with or without miRNA-binding sites were collectedafter 4 weeks and measured for total cholesterol, high-densitylipoprotein (HDL) and low-density lipoprotein (LDL). miR, microRNA;nLacZ, (3-galactosidase reporter transgene; rAAV, recombinantadeno-associated viruses.

FIGS. 15A to 15F depict a molecular characterization of transgene mRNAswith or without miRNA binding sites. (FIG. 15A) Locations of the probesand primers, the sequences of mature miR-122 and its perfectlycomplementary binding site in the transgene mRNA are presented. (FIG.15B) Total cellular RNA from liver was analyzed either by conventionalreverse-transcription PCR (RT-PCR) by using primers that span a regionbetween the 3′ end of nLacZ and the 5′ end of poly(A) signal (FIG. 15C)or by quantitative RT-PCR; data are presented as relative nLacZ mRNAlevels normalized to β-actin. (FIG. 15D) For the northern blot analysisof nLacZ mRNA, 18S RNA served as a loading control, and the blots werehybridized with either a transgene DNA (FIG. 15E) or RNA probe. (FIG.15F) In addition, poly(A) bearing mRNA from the liver of an animalreceived rAAV containing three miR-1- and three miR-122-binding siteswas analyzed by 5′ RACE; the PCR product was resolved on an ethidiumbromide-stained agarose gel. miR, microRNA; nLacZ, β-galactosidasereporter transgene; rAAV, recombinant adeno-associated viruses.

FIGS. 16A to 16B depict an alignment of sequences spanning themiRNA-binding sites and poly(A) signal regions recovered by 5 RACE.Poly(A)-containing mRNA was isolated from the (FIG. 16A) liver and (FIG.16B) heart of an animal injected withrAAV9CBnLacZ-(miR-1BS)₃-(miR-122BS)₃. Twenty-one liver-derived andtwenty-two heart-derived clones were sequenced. The putative cleavagesites in each clone are identified by arrows; the frequencies ofmiRNA-directed, site-specific cleavage for each miRNA-binding site arereported; triangles point to the positions of the expectedmiRNA-directed cleavage sites (FIGS. 16A to 16B). miRNA, microRNA,nLacZ, β-galactosidase reporter transgene; rAAV, recombinantadeno-associated viruses.

FIGS. 17A to 17B depict an endogenous miRNA-repressed, CNS-directed EGFPgene transfer by systemically delivered rAAV9. Ten-week-old male C57BL/6mice were injected intravenously with scAAV9CBEGFP orscAAV9CBnLacZ(miR-1BS)₃-(miR-122BS)₃ at a dose of 2×10¹⁴ genome copiesper kg (GC/kg) body weight. The animals were necropsied 3 weeks laterfor whole body fixation by transcardiac perfusion. (FIG. 17A) Brain,spinal cord, liver, heart, and muscle were harvested for cryosectioning,immunofluorescent staining for EGFP (brain and cervical spinal cord),and fluorescence microscopy to detect EGFP. Total cellular DNA and RNAwere extracted from brain, liver, heart and muscle to measure the amountof persistent vector genome by qPCR and EGFP mRNA by qRT-PCR. (FIG. 17B)For each tissue, the relative abundance of the EGFP mRNA containingmiRNA-binding sites was compared to that of the EGFP mRNA lackingmiRNA-binding sites. For each sample, mRNA abundance was normalized tothe amount of vector genome detected in the tissue. EGFP, enhanced greenfluorescent protein; miRNA, microRNA; nLacZ, β-galactosidase reportertransgene; qRT-PCR, quantitative reverse-transcription PCR; rAAV,recombinant adeno-associated viruses.

FIG. 18 depicts a molecular model for endogenous miRNA-regulated rAAVexpression. miRNA, microRNA; rAAV, recombinant adeno-associated viruses.

FIGS. 19A to 19D depict a quantification of GFP intensity levels in thebrain and spinal cord of neonatal mice transduced with various AAVvectors. 4×10¹¹ genome copies (GCs) of ten different AAV vectors wereinjected into neonatal P1 pups through superfacial vein. The mice weresacrificed 21 days after injection. The brain tissues were extracted and40 μm thick cryosections were prepared. The sections were stainedagainst anti-EGFP antibody. The images were analyzed and theintensity/pixel values of all AAV serotypes in various regions in brainand spinal cord (FIGS. 19A to 19C) were calculated by using Nikon NISelements AR software version 3.2. Average intensities of the brain andspinal cord regions for different rAAVs were also presented (FIG. 19D).Region of interest (ROI) of each anatomical structure was fixed for allvectors to ensure the parallel comparison.

FIG. 20 depicts a strong and widespread EGFP expression in neonatalmouse brain after intravenous injection of rAAVs. 4×10¹¹ genome copies(GCs) of rAAVs 7, 9, rh.10, rh.39 and rh.43 were injected into neonatalP1 pups through superfacial vein. The mice were sacrificed 21 days afterinjection. The brain tissues were extracted and 40 μm thick cryosectionswere prepared. The sections were stained against anti-EGFP antibody.Bars represent 100 μm. The regions shown are: olfactory bulb, striatum,hippocampus, cortex, hypothalamus, cerebellum and medulla.

FIG. 21 depicts EGFP expression in neonatal mouse spinal cord afterintravenous injection of rAAVs. 4×10¹¹ GCs of rAAVs 7, 9, rh.10, rh.39and rh.43 were injected into neonatal P1 pups through superfacial vein.The mice were sacrificed 21 days after injection. The spinal cordtissues were extracted and 40 μm thick cryosections were prepared. Thesections from cervical, thoracic and lumbar regions were stained againstanti-EGFP antibody. Bars represent 100 μm.

FIG. 22 depicts EGFP expression in dorsal root ganglia transduced byintravascularly delivered rAAVs1, 2, 6, 6.2, 7, 9, rh.10 and rh.39.Neonatal pups received 4×10¹¹ GCs of rAAVs at P1 and were necropsied 21days after injection. Forty μm thick cryosections were processed fordouble immunohistochemical staining for EGFP (green) and Neurons (NeuN,red). Bars represent 75 μm.

FIG. 23 depicts confocal microscopic analysis of the transduced celltypes in mouse CNS after systemic delivery of rAAVs to P1 neonates. The40 μm thick brain and spinal cord sections of the animals treated withdifferent rAAVs were co-strained against anti-EGFP antibody andcell-type specific markers. Anti-NeuN was used to stain neuronal cells;anti-GFAP was used to stain astrocytes; anti-Calbindin was used to stainPurkinje cells; anti-ChAT was used to stain motor neurons; anti-DARPPwas used to stain dopaminergic neurons in the substantia nigra. AllrAAVs were examined, but for each cell type, only one representativepicture was shown here.

FIG. 24 depicts a transduction of the brain ventricular structures byintravascularly delivered rAAVs. Neonatal pups received 4×10¹¹ GCs ofrAAVs at P1 and were necropsied 21 days after injection. The choroidplexuses in different ventricles were well preserved during tissueprocess. Forty μm thick cryosections were stained against anti-EGFPantibody. Bars represent 100 μm.

FIGS. 25A to 25B depict an analysis of purity and morphologicalintegrity of rAAV vectors. FIG. 25A. Silver stained SDS-Page analysis ofCsCl gradient purified rAAVCBEGFP vectors used in this study.Approximately 1.5×1010 virus particles each of rAAVs 1, 2, 5, 6, 6.2, 7,9, rh10, rh39 and rh43 were loaded in the corresponding lane. FIG. 25B.Transmission electron microscopy of negative stained recombinant AAVvirions. rAAV virions were spread on a freshly prepared carboncoated—Formvar support film and stained with 1% uranyl acetate fortransmission microscopy. The images of virus particles fromrepresentative vector lots were taken at 92,000× and presented.

FIG. 26 depicts a transduction of neonatal mouse dorsal root ganglia bysystemically delivered rAAVs 1, 6, 6.2 and rh43. Neonatal pups received4×10¹¹ GCs of rAAVs at P1 were necropsied 21 days after injection. Fortyμm thick cryosections were stained against anti-EGFP antibody. Barsrepresent 75 μm.

FIGS. 27A to 27B depict a transduction of the brain capillary vessels byintravascularly delivered rAAVs. Neonatal pups received 4×10¹¹ GCs ofrAAVs at P1 were necropsied 21 days after injection. Forty μm thickcryosections of the brains were stained against: (FIG. 27A) anti-EGFPantibody (AAV1, AAV6, AAV6.2, AAV7, AAV9, AAVrh.10, AAVrh.39 andAAVrh.43); (FIG. 27B) anti-EGFP and anti-CD34 antibodies (rh.10 only).Bars represent 100 μm.

FIG. 28 depicts an evaluation of microgliosis in mice brain aftersystemic delivery of rAAVs to P1 neonates. The 40 μm thick brainsections of the animals treated with different rAAVs were co-strainedagainst anti-EGFP antibody and anti-IBa-1. Only the staining result ofrAAVrh.10 was shown.

FIG. 29 depicts native EGFP expression in mice CNS after systemicdelivery of rAAVs to P1 neonates. Neonatal pups received 4×10¹¹ GCs ofrAAVs at P1 were necropsied 21 days after injection. Forty μm thickcryosections were mounted and observed under microscope withoutimmunostaining. The exposure times for each image were indicated.

FIGS. 30A to 30E depict results showing the effects of rAAV based genetherapy in the treatment of Canavan disease. FIG. 30A shows thattreatment corrected gait and motor function of the CD mice. FIG. 30Bshows that treatment mitigated retinopathy and restored vision in CDmice. FIG. 30C shows that NAA levels in the treated CD mice approachthose in the normal mice. FIG. 30D indicates that APSA activity isdetected in the brains of CD mice. FIG. 30E indicates APSA expression isdetected in the brains of CD mice.

FIG. 31A depicts that vacuolation in both brain and spinal cord of thetreated mice is more patchy and variable with generally smaller-sizedvacuoles and that some areas of the cerebral cortex show almost novacuolation. FIG. 31B shows ASPA expression in the cerebral cortex insitu.

FIGS. 32A to 32B depict results of a quantitative analysis ofvacuolation in various brain regions. FIG. 32A shows that olfactory bulbhad a dramatic mitigation in the white matter degeneration after genetherapy and that the large vacuoles were essentially eliminated in othertissues. FIG. 32B shows results from a similar analysis on spinal cordsections.

FIGS. 33A to 33D depict results from a histopathological evaluation ofkidneys in the CD mice. FIG. 33A shows that the renal tubular epitheliumof the kidney was diffusely attenuated and exhibited enlargement of thetubular lumens in untreated CD mice. FIG. 33B shows that treated CDmouse had normal glomeruli. FIGS. 33C and 33D depict results of ananalysis of two lead candidate vectors, rAAV9 and rh.10, respectively,for efficiency of kidney transduction after IV delivery.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Adeno-associated virus (AAV) is a small (26 nm) replication-defective,nonenveloped virus, that depends on the presence of a second virus, suchas adenovirus or herpes virus, for its growth in cells. AAV is not knownto cause disease and induces a very mild immune response. AAV can infectboth dividing and non-dividing cells and may incorporate its genome intothat of the host cell. Aspects of the invention provide methods fordelivering a transgene to a CNS tissue in a subject using recombinantAAV-based gene transfer. Accordingly, methods and compositions fortreating CNS-related disorders are provided herein. Further aspects ofthe invention, are based on the discovery of rAAVs that achievewide-spread distribution throughout CNS tissue. In some embodiments, therAAVs spread throughout CNS tissue following direct administration intothe cerebrospinal fluid (CSF), e.g., via intrathecal and/orintracerebral injection. In other embodiments, the rAAVs cross theblood-brain-barrier and achieve wide-spread distribution throughout CNStissue of a subject following intravenous administration. Such rAAVs areuseful for the treatment of CNS-related disorders, including, forexample, amyotrophic lateral sclerosis (ALS) and Canavan disease (CD).

Methods and Compositions for Targeting CNS Tissue

Methods for delivering a transgene to central nervous system (CNS)tissue in a subject are provided herein. The methods typically involveadministering to a subject an effective amount of a rAAV comprising anucleic acid vector for expressing a transgene in the subject. An“effective amount” of a rAAV is an amount sufficient to infect asufficient number of cells of a target tissue in a subject. An effectiveamount of a rAAV may be an amount sufficient to have a therapeuticbenefit in a subject, e.g., to extend the lifespan of a subject, toimprove in the subject one or more symptoms of disease, e.g., a symptomof ALS, a symptom of Canavan disease, etc. In some cases, an effectiveamount of a rAAV may be an amount sufficient to produce a stable somatictransgenic animal model. The effective amount will depend on a varietyof factors such as, for example, the species, age, weight, health of thesubject, and the CNS tissue to be targeted, and may thus vary amongsubject and tissue. An effective amount may also depend on the mode ofadministration. For example, targeting a CNS tissue by intravascularinjection may require different (e.g., higher) doses, in some cases,than targeting CNS tissue by intrathecal or intracerebral injection. Insome cases, multiple doses of a rAAV are administered. An effectiveamount may also depend on the rAAV used. For example, dosages fortargeting a CNS tissue may depend on the serotype (e.g., the capsidprotein) of the rAAV. For example, the rAAV may have a capsid protein ofa AAV serotype selected from the group consisting of: AAV1, AAV2, AAV5,AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43 and CSp3. In certainembodiments, the effective amount of rAAV is 10¹⁰, 10¹¹, 10¹², 10¹³, or10¹⁴ genome copies per kg. In certain embodiments, the effective amountof rAAV is 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ genome copies persubject.

A method for delivering a transgene to CNS tissue in a subject maycomprise administering a rAAV by a single route or by multiple routes.For example, delivering a transgene to CNS tissue in a subject maycomprise administering to the subject, by intravenous administration, aneffective amount of a rAAV that crosses the blood-brain-barrier.Delivering a transgene to CNS tissue in a subject may compriseadministering to the subject an effective amount of a rAAV byintrathecal administration or intracerebral administration, e.g., byintraventricular injection. A method for delivering a transgene to CNStissue in a subject may comprise co-administering of an effective amountof a rAAV by two different administration routes, e.g., by intrathecaladministration and by intracerebral administration. Co-administrationmay be performed at approximately the same time, or different times.

The CNS tissue to be targeted may be selected from cortex, hippocampus,thalamus, hypothalamus, cerebellum, brain stem, cervical spinal cord,thoracic spinal cord, and lumbar spinal cord, for example. Theadministration route for targeting CNS tissue typically depends on theAAV serotype. For example, in certain instances where the AAV serotypeis selected from AAV1, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10, rh.39,rh.43 and CSp3, the administration route may be intravascular injection.In some instances, for example where the AAV serotype is selected fromAAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43and CSp3, the administration route may be intrathecal and/orintracerebral injection.

Intravascular Administration

As used herein the term “intravascular administration” refers to theadministration of an agent, e.g., a composition comprising a rAAV, intothe vasculature of a subject, including the venous and arterialcirculatory systems of the subject. Typically, rAAVs that cross theblood-brain-barrier may be delivered by intravascular administration fortargeting CNS tissue. In some cases, intravascular (e.g., intravenous)administration facilitates the use of larger volumes than other forms ofadministration (e.g., intrathecal, intracerebral). Thus, large doses ofrAAVs (e.g., up to 10¹⁵ GC/subject) can be delivered at one time byintravascular (e.g., intravenous) administration. Methods forintravascular administration are well known in the art and include forexample, use of a hypodermic needle, peripheral cannula, central venousline, etc.

Intrathecal and/or Intracerebral Administration

As used herein the term “intrathecal administration” refers to theadministration of an agent, e.g., a composition comprising a rAAV, intothe spinal canal. For example, intrathecal administration may compriseinjection in the cervical region of the spinal canal, in the thoracicregion of the spinal canal, or in the lumbar region of the spinal canal.Typically, intrathecal administration is performed by injecting anagent, e.g., a composition comprising a rAAV, into the subarachnoidcavity (subarachnoid space) of the spinal canal, which is the regionbetween the arachnoid membrane and pia mater of the spinal canal. Thesubarchnoid space is occupied by spongy tissue consisting of trabeculæ(delicate connective tissue filaments that extend from the arachnoidmater and blend into the pia mater) and intercommunicating channels inwhich the cerebrospinal fluid is contained. In some embodiments,intrathecal administration is not administration into the spinalvasculature.

As used herein, the term “intracerebral administration” refers toadministration of an agent into and/or around the brain. Intracerebraladministration includes, but is not limited to, administration of anagent into the cerebrum, medulla, pons, cerebellum, intracranial cavity,and meninges surrounding the brain. Intracerebral administration mayinclude administration into the dura mater, arachnoid mater, and piamater of the brain. Intracerebral administration may include, in someembodiments, administration of an agent into the cerebrospinal fluid(CSF) of the subarachnoid space surrounding the brain. Intracerebraladministration may include, in some embodiments, administration of anagent into ventricles of the brain, e.g., the right lateral ventricle,the left lateral ventricle, the third ventricle, the fourth ventricle.In some embodiments, intracerebral administration is not administrationinto the brain vasculature.

Intracerebral administration may involve direct injection into and/oraround the brain. In some embodiments, intracerebral administrationinvolves injection using stereotaxic procedures. Stereotaxic proceduresare well know in the art and typically involve the use of a computer anda 3-dimensional scanning device that are used together to guideinjection to a particular intracerebral region, e.g., a ventricularregion. Micro-injection pumps (e.g., from World Precision Instruments)may also be used. In some embodiments, a microinjection pump is used todeliver a composition comprising a rAAV. In some embodiments, theinfusion rate of the composition is in a range of 1 μl/minute to 100μl/minute. As will be appreciated by the skilled artisan, infusion rateswill depend on a variety of factors, including, for example, species ofthe subject, age of the subject, weight/size of the subject, serotype ofthe AAV, dosage required, intracerebral region targeted, etc. Thus,other infusion rates may be deemed by a skilled artisan to beappropriate in certain circumstances.

Methods and Compositions for Treating CNS-Related Disorders

Methods and compositions for treating CNS-related disorders are alsoprovided herein. As used herein, a “CNS-related disorder” is a diseaseor condition of the central nervous system. A CNS-related disorder mayaffect the spinal cord (e.g., a myelopathy), brain (e.g., aencephalopathy) or tissues surrounding the brain and spinal cord. ACNS-related disorder may be of a genetic origin, either inherited oracquired through a somatic mutation. A CNS-related disorder may be apsychological condition or disorder, e.g., Attention DeficientHyperactivity Disorder, Autism Spectrum Disorder, Mood Disorder,Schizophrenia, Depression, Rett Syndrome, etc. A CNS-related disordermay be an autoimmune disorder. A CNS-related disorder may also be acancer of the CNS, e.g., brain cancer. A CNS-related disorder that is acancer may be a primary cancer of the CNS, e.g., an astrocytoma,glioblastomas, etc., or may be a cancer that has metastasized to CNStissue, e.g., a lung cancer that has metastasized to the brain. Furthernon-limiting examples of CNS-related disorders, include Parkinson'sDisease, Lysosomal Storage Disease, Ischemia, Neuropathic Pain,Amyotrophic lateral sclerosis (ALS), Multiple Sclerosis (MS), andCanavan disease (CD).

Methods for treating amyotrophic lateral sclerosis (ALS) in a subject inneed thereof are provided herein. A subject in need of a treatment forALS is a subject having or suspected of having ALS. In some cases, ALShas been linked to a mutation in the gene coding for superoxidedismutase (SOD1). Elevated levels of SOD1 appear to be associated withALS in some instances. It has been shown that transgenic expression ofshRNA against SOD1 can knockdown mutant SOD1 expression, delay diseaseonset and extend survival (Xia et al. 2006, Neurobiol Dis 23: 578).Intrathecal infusion of siRNA against SOD1 at disease onset has alsobeen found to knockdown mutant SOD1 expression and extend survival (Wanget al. 2008, JBC 283: 15845). Furthermore, nerve injection of adenovirusexpressing shRNA against SOD1 at the disease onset can knockdown mutantSOD1 expression and extend survival (Wu et al. 2009, Antiox Redox Sig11: 1523).

Aspects of the invention, are based on the discovery of AAV-basedtherapies that achieve, with low-toxicity, long-term inhibition of SOD1expression that is wide-spread throughout CNS tissue of the subject.Methods for treating ALS that are provided herein, typically involveadministering to CNS tissue of a subject an effective amount of a rAAVthat harbors a nucleic acid comprising a promoter operably linked with aregion encoding an inhibitory RNA that binds specifically to SOD1 mRNA(e.g., that hybridizes specifically to a nucleic acid having a sequenceas set forth in SEQ ID NO 17 or 19) and inhibits expression of SOD1 inthe subject. It has been discovered that rAAVs having a capsid proteincomprising a sequence as set forth in SEQ ID NO: 9 achieve wide-spreaddistribution throughout the CNS following intrathecal injection and/orintracerebral injection, and thus, are particularly useful for treatingALS. This result is surprising in light of certain other rAAVs thatinfect cells only within the immediate vicinity of the injection site,or the achieve only a limited distribution, following intrathecalinjection. Thus, rAAVs that achieve wide-spread distribution throughoutthe CNS are particularly useful as gene transfer vectors for treatingALS.

In some embodiments, it has been discovered that co-administration byintrathecal injection and intracerebral injection, e.g.,intraventricular injection, of rAAVs having a capsid protein comprisinga sequence as set forth in SEQ ID NO: 9 and a nucleic acid comprising apromoter operably linked with a region encoding an inhibitory RNA thatbinds specifically to SOD1 mRNA and inhibits expression of SOD1,achieves long-term inhibition of SOD1 and improves outcome (e.g.,lifespan) in an animal model of ALS (See, e.g., FIG. 6A). In someembodiments, the inhibitory RNA is an antisense RNA, a shRNA or a miRNA.The inhibitory RNA may have a sequence as set forth in SEQ ID NO: 26.The inhibitory RNA may have a sequence as set forth in any one of SEQ IDNO: 22 to 30. Thus, in some embodiments, a nucleic acid comprising apromoter operably linked with a nucleic acid having a sequence as setforth in any one of SEQ ID NO: 22 to 30 is provided. In someembodiments, a recombinant AAV that harbors a nucleic acid comprising asequence as set forth in any one of SEQ ID NO: 22 to 30 is provided. Therecombinant AAV may have a capsid protein comprising a sequence as setforth in SEQ ID NO: 9. The recombinant AAV may have a capsid proteincomprising a sequence as set forth in any one of SEQ ID NO: 1 to 12.

Methods for treating Canavan disease (CD) in a subject in need thereofare provided herein. A subject in need of a treatment for CD is asubject having or suspected of having CD. Canavan disease is caused by adefective ASPA gene which is responsible for the production of theenzyme aspartoacylase. This enzyme normally breaks down the concentratedbrain molecule N-acetyl aspartate. Decreased aspartoacylase activity insubjects with CD prevents the normal breakdown of N-acetyl aspartate,and the lack of breakdown appears to interfere with growth of the myelinsheath of the nerve fibers in the brain. Symptoms of Canavan disease,which may appear in early infancy and progress rapidly, may includemental retardation, loss of previously acquired motor skills, feedingdifficulties, abnormal muscle tone (i.e., floppiness or stiffness), poorhead control, and megalocephaly (abnormally enlarged head). Paralysis,blindness, or seizures may also occur. Aspects of the invention improveone or more symptoms of CD in a subject by administering to the subjecta recombinant AAV harboring a nucleic acid that expresses aspartoacylase(ASPA). For example, a method for treating Canavan disease in a subjectin need thereof may comprise administering an effective amount of a rAAVto CNS tissue of the subject by intravascular administration, whereinthe rAAV comprises a nucleic acid comprising a promoter operably linkedwith a region encoding ASPA (e.g., a region having a sequence as setforth in SEQ ID NO: 14 or 16). A method for treating Canavan disease ina subject in need thereof may comprise administering an effective amountof a rAAV to CNS tissue of the subject by intrathecal administration,wherein the rAAV comprises a nucleic acid comprising a promoter operablylinked with a region encoding ASPA. In some cases, methods for treatingCD involve administering, to CNS tissue of the subject, an effectiveamount of a rAAV that comprises a capsid protein other than a capsidprotein of AAV serotype 2 (e.g., other than a protein having an aminoacid sequence as set forth in SEQ ID NO: 2) and a nucleic acidcomprising a promoter operably linked with a region encoding ASPA. Inanother example, a method for treating Canavan disease in a subject inneed thereof comprises administering an effective amount of a rAAV toCNS tissue of the subject by a route other than intracerebraladministration, wherein the rAAV comprises a nucleic acid comprising apromoter operably linked with a region encoding ASPA. In someembodiments, ASPA expressed in CNS tissue following administration ofthe rAAV results in a decrease in aspartoacylase activity and breakdownof N-acetyl aspartate in the CNS tissue. Thus, in some embodiments, arecombinant AAV vector is provided that comprises a nucleic acidencoding a sequence as set forth in SEQ ID NO: 14 or 16. In someembodiments, a recombinant AAV is provided that harbors a nucleic acidcomprising a promoter operably linked with a region having a sequence asset forth in SEQ ID NO: 14 or 16. In some embodiments, a recombinant AAVis provided that harbors a nucleic acid comprising a promoter operablylinked with a region encoding a protein having a sequence as set forthin SEQ ID NO: 13 or 15. The recombinant AAV may have a capsid proteincomprising an amino acid sequence as set forth in any one of SEQ ID NO:1 to 12. The recombinant AAV may have a capsid protein comprising asequence as set forth in any one of SEQ ID NO: 1 and 3 to 12.

Recombinant AAVs

In some aspects, the invention provides isolated AAVs. As used hereinwith respect to AAVs, the term “isolated” refers to an AAV that has beenisolated from its natural environment (e.g., from a host cell, tissue,or subject) or artificially produced. Isolated AAVs may be producedusing recombinant methods. Such AAVs are referred to herein as“recombinant AAVs”. Recombinant AAVs (rAAVs) preferably havetissue-specific targeting capabilities, such that a transgene of therAAV will be delivered specifically to one or more predeterminedtissue(s). The AAV capsid is an important element in determining thesetissue-specific targeting capabilities. Thus, a rAAV having a capsidappropriate for the tissue being targeted can be selected. In someembodiments, the rAAV comprises a capsid protein having an amino acidsequence as set forth in any one of SEQ ID NOs 1 to 12, or a proteinhaving substantial homology thereto.

Methods for obtaining recombinant AAVs having a desired capsid proteinare well known in the art (See, for example, US 2003/0138772, thecontents of which are incorporated herein by reference in theirentirety). AAVs capsid protein that may be used in the rAAVs of theinvention a include, for example, those disclosed in G. Gao, et al., J.Virol, 78(12):6381-6388 (June 2004); G. Gao, et al, Proc Natl Acad SciUSA, 100(10):6081-6086 (May 13, 2003); US 2003-0138772, US 2007/0036760,US 2009/0197338, and U.S. provisional application Ser. No. 61/182,084,filed May 28, 2009, the contents of which relating to AAVs capsidproteins and associated nucleotide and amino acid sequences areincorporated herein by reference. Typically the methods involveculturing a host cell which contains a nucleic acid sequence encoding anAAV capsid protein (e.g., a nucleic acid encoding a protein having asequence as set forth in any one of SEQ ID NOs 1-12) or fragmentthereof; a functional rep gene; a recombinant AAV vector composed of,AAV inverted terminal repeats (ITRs) and a transgene; and sufficienthelper functions to permit packaging of the recombinant AAV vector intothe AAV capsid proteins.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein, in the discussion ofregulatory elements suitable for use with the transgene. In stillanother alternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the invention may bedelivered to the packaging host cell using any appropriate geneticelement (vector). The selected genetic element may be delivered by anysuitable method, including those described herein. The methods used toconstruct any embodiment of this invention are known to those with skillin nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Sambrooket al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virionsare well known and the selection of a suitable method is not alimitation on the present invention. See, e.g., K. Fisher et al, J.Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the tripletransfection method (e.g., as described in detail in U.S. Pat. No.6,001,650, the contents of which relating to the triple transfectionmethod are incorporated herein by reference). Typically, the recombinantAAVs are produced by transfecting a host cell with a recombinant AAVvector (comprising a transgene) to be packaged into AAV particles, anAAV helper function vector, and an accessory function vector. An AAVhelper function vector encodes the “AAV helper function” sequences(i.e., rep and cap), which function in trans for productive AAVreplication and encapsidation. Preferably, the AAV helper functionvector supports efficient AAV vector production without generating anydetectable wild-type AAV virions (i.e., AAV virions containingfunctional rep and cap genes). Non-limiting examples of vectors suitablefor use with the present invention include pHLP19, described in U.S.Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No.6,156,303, the entirety of both incorporated by reference herein. Theaccessory function vector encodes nucleotide sequences for non-AAVderived viral and/or cellular functions upon which AAV is dependent forreplication (i.e., “accessory functions”). The accessory functionsinclude those functions required for AAV replication, including, withoutlimitation, those moieties involved in activation of AAV genetranscription, stage specific AAV mRNA splicing, AAV DNA replication,synthesis of cap expression products, and AAV capsid assembly.Viral-based accessory functions can be derived from any of the knownhelper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1), and vaccinia virus.

In some aspects, the invention provides transfected host cells. The term“transfection” is used to refer to the uptake of foreign DNA by a cell,and a cell has been “transfected” when exogenous DNA has been introducedinside the cell membrane. A number of transfection techniques aregenerally known in the art. See, e.g., Graham et al. (1973) Virology,52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual,Cold Spring Harbor Laboratories, New York, Davis et al. (1986) BasicMethods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousnucleic acids, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable ofharboring, a substance of interest. Often a host cell is a mammaliancell. A host cell may be used as a recipient of an AAV helper construct,an AAV minigene plasmid, an accessory function vector, or other transferDNA associated with the production of recombinant AAVs. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein may refer to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

In some aspects, the invention provides isolated cells. As used hereinwith respect to cell, the term “isolated” refers to a cell that has beenisolated from its natural environment (e.g., from a tissue or subject).As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants. As used herein, the terms“recombinant cell” refers to a cell into which an exogenous DNA segment,such as DNA segment that leads to the transcription of abiologically-active polypeptide or production of a biologically activenucleic acid such as an RNA, has been introduced.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrases“operatively positioned,” “under control” or “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene. The term “expression vector orconstruct” means any type of genetic construct containing a nucleic acidin which part or all of the nucleic acid encoding sequence is capable ofbeing transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically-active polypeptide product or inhibitory RNA (e.g., shRNA,miRNA) from a transcribed gene.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs of the invention are not meant to belimiting and other suitable methods will be apparent to the skilledartisan.

Recombinant AAV Vectors

“Recombinant AAV (rAAV) vectors” of the invention are typically composedof, at a minimum, a transgene and its regulatory sequences, and 5′ and3′ AAV inverted terminal repeats (ITRs). It is this recombinant AAVvector which is packaged into a capsid protein and delivered to aselected target cell. In some embodiments, the transgene is a nucleicacid sequence, heterologous to the vector sequences, which encodes apolypeptide, protein, functional RNA molecule (e.g., miRNA, miRNAinhibitor) or other gene product, of interest. The nucleic acid codingsequence is operatively linked to regulatory components in a mannerwhich permits transgene transcription, translation, and/or expression ina cell of a target tissue.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. (See, e.g., texts such as Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Anexample of such a molecule employed in the present invention is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified mammalian AAV types.

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elements whichare operably linked to the transgene in a manner which permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by the invention.As used herein, “operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest. Expression control sequences includeappropriate transcription initiation, termination, promoter and enhancersequences; efficient RNA processing signals such as splicing andpolyadenylation (polyA) signals; sequences that stabilize cytoplasmicmRNA; sequences that enhance translation efficiency (i.e., Kozakconsensus sequence); sequences that enhance protein stability; and whendesired, sequences that enhance secretion of the encoded product. Agreat number of expression control sequences, including promoters whichare native, constitutive, inducible and/or tissue-specific, are known inthe art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be operably linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably linked to a nucleic acidsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide. Similarly two ormore coding regions are operably linked when they are linked in such away that their transcription from a common promoter results in theexpression of two or more proteins having been translated in frame. Insome embodiments, operably linked coding sequences yield a fusionprotein. In some embodiments, operably linked coding sequences yield afunctional RNA (e.g., shRNA, miRNA).

For nucleic acids encoding proteins, a polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ AAV ITR sequence. A rAAV construct useful in the present inventionmay also contain an intron, desirably located between thepromoter/enhancer sequence and the transgene. One possible intronsequence is derived from SV-40, and is referred to as the SV-40 T intronsequence. Another vector element that may be used is an internalribosome entry site (IRES). An IRES sequence is used to produce morethan one polypeptide from a single gene transcript. An IRES sequencewould be used to produce a protein that contain more than onepolypeptide chains. Selection of these and other common vector elementsare conventional and many such sequences are available [see, e.g.,Sambrook et al, and references cited therein at, for example, pages 3.183.26 and 16.17 16.27 and Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1989]. In some embodiments, a Footand Mouth Disease Virus 2A sequence is included in polyprotein; this isa small peptide (approximately 18 amino acids in length) that has beenshown to mediate the cleavage of polyproteins (Ryan, M D et al., EMBO,1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin,C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity ofthe 2A sequence has previously been demonstrated in artificial systemsincluding plasmids and gene therapy vectors (AAV and retroviruses)(Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M et al., JVirology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy,2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe,P et al., Human Gene Therapy, 2000; 11: 1921-1931.; and Klump, H et al.,Gene Therapy, 2001; 8: 811-817).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of theinvention may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), thetetracycline-inducible system (Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)), the RU486-inducible system (Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and therapamycin-inducible system (Magari et al, J. Clin. Invest.,100:2865-2872 (1997)). Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

In another embodiment, the native promoter, or fragment thereof, for thetransgene will be used. The native promoter may be preferred when it isdesired that expression of the transgene should mimic the nativeexpression. The native promoter may be used when expression of thetransgene must be regulated temporally or developmentally, or in atissue-specific manner, or in response to specific transcriptionalstimuli. In a further embodiment, other native expression controlelements, such as enhancer elements, polyadenylation sites or Kozakconsensus sequences may also be used to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art. Exemplary tissue-specific regulatory sequences include, butare not limited to the following tissue specific promoters: neuronalsuch as neuron-specific enolase (NSE) promoter (Andersen et al., Cell.Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain genepromoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5(1991)), and the neuron-specific vgf gene promoter (Piccioli et al.,Neuron, 15:373-84 (1995)). In some embodiments, the tissue-specificpromoter is a promoter of a gene selected from: neuronal nuclei (NeuN),glial fibrillary acidic protein (GFAP), adenomatous polyposis coli(APC), and ionized calcium-binding adapter molecule 1 (Iba-1). Otherappropriate tissue specific promoters will be apparent to the skilledartisan. In some embodiments, the promoter is a chicken Beta-actinpromoter.

In some embodiments, one or more bindings sites for one or more ofmiRNAs are incorporated in a transgene of a rAAV vector, to inhibit theexpression of the transgene in one or more tissues of a subjectharboring the transgenes, e.g., non-CNS tissues. The skilled artisanwill appreciate that binding sites may be selected to control theexpression of a transgene in a tissue specific manner. For example,expression of a transgene in the liver may be inhibited by incorporatinga binding site for miR-122 such that mRNA expressed from the transgenebinds to and is inhibited by miR-122 in the liver. Expression of atransgene in the heart may be inhibited by incorporating a binding sitefor miR-133a or miR-1, such that mRNA expressed from the transgene bindsto and is inhibited by miR-133a or miR-1 in the heart. The miRNA targetsites in the mRNA may be in the 5′ UTR, the 3′ UTR or in the codingregion. Typically, the target site is in the 3′ UTR of the mRNA.Furthermore, the transgene may be designed such that multiple miRNAsregulate the mRNA by recognizing the same or multiple sites. Thepresence of multiple miRNA binding sites may result in the cooperativeaction of multiple RISCs and provide highly efficient inhibition ofexpression. The target site sequence may comprise a total of 5-100,10-60, or more nucleotides. The target site sequence may comprise atleast 5 nucleotides of the sequence of a target gene binding site.

Transgene Coding Sequences: CNS-Related Genes

The composition of the transgene sequence of a rAAV vector will dependupon the use to which the resulting vector will be put. For example, onetype of transgene sequence includes a reporter sequence, which uponexpression produces a detectable signal. In another example, thetransgene encodes a therapeutic protein or therapeutic functional RNA.In another example, the transgene encodes a protein or functional RNAthat is intended to be used for research purposes, e.g., to create asomatic transgenic animal model harboring the transgene, e.g., to studythe function of the transgene product. In another example, the transgeneencodes a protein or functional RNA that is intended to be used tocreate an animal model of disease. Appropriate transgene codingsequences will be apparent to the skilled artisan.

In some aspects, the invention provides rAAV vectors for use in methodsof preventing or treating one or more gene defects (e.g., heritable genedefects, somatic gene alterations) in a mammal, such as for example, agene defect that results in a polypeptide deficiency or polypeptideexcess in a subject, and particularly for treating or reducing theseverity or extent of deficiency in a subject manifesting aCNS-associated disorder linked to a deficiency in such polypeptides incells and tissues. In some embodiments, methods involve administrationof a rAAV vector that encodes one or more therapeutic peptides,polypeptides, shRNAs, microRNAs, antisense nucleotides, etc. in apharmaceutically-acceptable carrier to the subject in an amount and fora period of time sufficient to treat the CNS-associated disorder in thesubject having or suspected of having such a disorder.

A rAAV vector may comprise as a transgene, a nucleic acid encoding aprotein or functional RNA that modulates or treats a CNS-associateddisorder. The following is a non-limiting list of genes associated withCNS-associated disorders: neuronal apoptosis inhibitory protein (NAIP),nerve growth factor (NGF), glial-derived growth factor (GDNF),brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF),tyrosine hydroxlase (TH), GTP-cyclohydrolase (GTPCH), aspartoacylase(ASPA), superoxide dismutase (SOD1) and amino acid decorboxylase (AADC).For example, a useful transgene in the treatment of Parkinson's diseaseencodes TH, which is a rate limiting enzyme in the synthesis ofdopamine. A transgene encoding GTPCH, which generates the TH cofactortetrahydrobiopterin, may also be used in the treatment of Parkinson'sdisease. A transgene encoding GDNF or BDNF, or AADC, which facilitatesconversion of L-Dopa to DA, may also be used for the treatment ofParkinson's disease. For the treatment of ALS, a useful transgene mayencode: GDNF, BDNF or CNTF. Also for the treatment of ALS, a usefultransgene may encode a functional RNA, e.g., shRNA, miRNA, that inhibitsthe expression of SOD1. For the treatment of ischemia a useful transgenemay encode NAIP or NGF. A transgene encoding Beta-glucuronidase (GUS)may be useful for the treatment of certain lysosomal storage diseases(e.g., Mucopolysacharidosis type VII (MPS VII)). A transgene encoding aprodrug activation gene, e.g., HSV-Thymidine kinase which convertsganciclovir to a toxic nucleotide which disrupts DNA synthesis and leadsto cell death, may be useful for treating certain cancers, e.g., whenadministered in combination with the prodrug. A transgene encoding anendogenous opioid, such a β-endorphin may be useful for treating pain.Other examples of transgenes that may be used in the rAAV vectors of theinvention will be apparent to the skilled artisan (See, e.g., CostantiniL C, et al., Gene Therapy (2000) 7, 93-109).

In some embodiments, the cloning capacity of the recombinant RNA vectormay be limited and a desired coding sequence may involve the completereplacement of the virus's 4.8 kilobase genome. Large genes may,therefore, not be suitable for use in a standard recombinant AAV vector,in some cases. The skilled artisan will appreciate that options areavailable in the art for overcoming a limited coding capacity. Forexample, the AAV ITRs of two genomes can anneal to form head to tailconcatamers, almost doubling the capacity of the vector. Insertion ofsplice sites allows for the removal of the ITRs from the transcript.Other options for overcoming a limited cloning capacity will be apparentto the skilled artisan.

Recombinant AAV Administration

rAAVS are administered in sufficient amounts to transfect the cells of adesired tissue and to provide sufficient levels of gene transfer andexpression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected tissue (e.g.,intracerebral administration, intrathecal administration), intravenous,oral, inhalation (including intranasal and intratracheal delivery),intraocular, intravenous, intramuscular, subcutaneous, intradermal,intratumoral, and other parental routes of administration. Routes ofadministration may be combined, if desired.

Delivery of certain rAAVs to a subject may be, for example, byadministration into the bloodstream of the subject. Administration intothe bloodstream may be by injection into a vein, an artery, or any othervascular conduit. Moreover, in certain instances, it may be desirable todeliver the rAAVs to brain tissue, meninges, neuronal cells, glialcells, astrocytes, oligodendrocytes, cereobrospinal fluid (CSF),interstitial spaces and the like. In some embodiments, recombinant AAVsmay be delivered directly to the spinal cord or brain by injection intothe ventricular region, as well as to the striatum (e.g., the caudatenucleus or putamen of the striatum), and neuromuscular junction, orcerebellar lobule, with a needle, catheter or related device, usingneurosurgical techniques known in the art, such as by stereotacticinjection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidsonet al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223,1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). Incertain circumstances it will be desirable to deliver the rAAV-basedtherapeutic constructs in suitably formulated pharmaceuticalcompositions disclosed herein either subcutaneously,intrapancreatically, intranasally, parenterally, intravenously,intramuscularly, intracerebrally, intrathecally, intracerebrally,orally, intraperitoneally, or by inhalation. In some embodiments, theadministration modalities as described in U.S. Pat. Nos. 5,543,158;5,641,515 and 5,399,363 (each specifically incorporated herein byreference in its entirety) may be used to deliver rAAVs.

Recombinant AAV Compositions

The rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. The rAAV, preferably suspended ina physiologically compatible carrier (e.g., in a composition), may beadministered to a subject, e.g., a human, mouse, rat, cat, dog, sheep,rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, ora non-human primate (e.g, Macaque). The compositions of the inventionmay comprise a rAAV alone, or in combination with one or more otherviruses (e.g., a second rAAV encoding having one or more differenttransgenes). In some embodiments, a compositions comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more different rAAVs each having one or moredifferent transgenes.

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the rAAV is directed. For example, onesuitable carrier includes saline, which may be formulated with a varietyof buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, andwater. The selection of the carrier is not a limitation of the presentinvention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The dose of rAAV virions required to achieve a desired effect or“therapeutic effect,” e.g., the units of dose in vector genomes/perkilogram of body weight (vg/kg), will vary based on several factorsincluding, but not limited to: the route of rAAV administration, thelevel of gene or RNA expression required to achieve a therapeuticeffect, the specific disease or disorder being treated, and thestability of the gene or RNA product. One of skill in the art canreadily determine a rAAV virion dose range to treat a subject having aparticular disease or disorder based on the aforementioned factors, aswell as other factors that are well known in the art. An effectiveamount of the rAAV is generally in the range of from about 10 μl toabout 100 ml of solution containing from about 10⁹ to 10¹⁶ genome copiesper subject. Other volumes of solution may be used. The volume used willtypically depend, among other things, on the size of the subject, thedose of the rAAV, and the route of administration. For example, forintrathecal or intracerebral administration a volume in range of 1 μl to10 μl or 10 μl to 100 μl may be used. For intravenous administration avolume in range of 10 μl to 100 μl, 100 μl to 1 ml, 1 ml to 10 ml, ormore may be used. In some cases, a dosage between about 10¹⁰ to 10¹²rAAV genome copies per subject is appropriate. In certain embodiments,10¹² rAAV genome copies per subject is effective to target CNS tissues.In some embodiments the rAAV is administered at a dose of 10¹⁰, 10¹¹,10¹², 10¹³, 10¹⁴, or 10¹⁵ genome copies per subject. In some embodimentsthe rAAV is administered at a dose of 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴genome copies per kg.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., ˜10¹³ GC/ml or more). Methods forreducing aggregation of rAAVs are well known in the art and, include,for example, addition of surfactants, pH adjustment, salt concentrationadjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy(2005) 12, 171-178, the contents of which are incorporated herein byreference.)

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens. Typically, these formulations may contain at least about 0.1%of the active ingredient or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active ingredient ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activerAAV in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The rAAV compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present invention intosuitable host cells. In particular, the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trails examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 .ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV compositions to a host. Sonophoresis (ie., ultrasound) has beenused and described in U.S. Pat. No. 5,656,016 as a device for enhancingthe rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

Kits and Related Compositions

The agents described herein may, in some embodiments, be assembled intopharmaceutical or diagnostic or research kits to facilitate their use intherapeutic, diagnostic or research applications. A kit may include oneor more containers housing the components of the invention andinstructions for use. Specifically, such kits may include one or moreagents described herein, along with instructions describing the intendedapplication and the proper use of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents. Kits for research purposes may contain the components inappropriate concentrations or quantities for running variousexperiments.

The kit may be designed to facilitate use of the methods describedherein by researchers and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for animal administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to asubject, such as a syringe, topical application devices, or IV needletubing and bag.

Examples Example 1: Characterization of 12 AAV Vectors for IntravascularDelivery to Target CNS and Detarget Non-CNS Tissues by miRNA Regulation

The CNS gene transfer properties of 12 scAAVEGFP vectors of differentserotypes, or natural variants were evaluated. RAAVs that cross theblood-brain-barrier (BBB) and target oligodendrocytes were discovered.Experiments were performed in neonatal mice (1 day old) and in adultmice (10 week old) (C57BL/6). The following AAV serotypes were tested:AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10 (also referredto herein as AAVrh.10), rh.39, rh.43, CSp3.

The recombinant AAV vectors expressed an enhanced GFP reporter geneunder the CMV-enhanced chicken β-actin hybrid promoter and were producedby transient transfection in 293 cells. The neonatal day 1 pups wereanesthetized with isoflurane. Then 100 μL of rAAV vectors (4×10¹¹ GC permouse) was injected to the pups via superfacial temporal vein under adissection microscope. In adult mice, rAAV was administered by tail veininjection (two different doses were evaluated 4×10¹¹ GC per mouse or4×10¹² GC per mouse). Twenty-one days post injection, the treatedanimals were anesthetized and transcardially perfused with cold PBS and4% (v/v) paraformaldehyde. Brains were extracted, immersed in 20%sucrose, and embedded in Tissue-Tek OCT. 40 μm thick sections were cutand stained in 12-well plate with primary antibodies, e.g., anti-NeuN,anti-EGFP and anti-GFAP, overnight at 4° C., then with secondaryantibodies for 2 h at room temperature. Control mice received PBSinjections.

In the neonatal study, the distribution of EGFP (+) cells throughout thebrain at 3 wks post-infusion was observed. Large numbers of EGFP (+)cells with variable intensities were visible in different regions of thebrains from the animals treated with 10 out of 12 vectors. In manyinstances the choroid plexus showed very strong EGFP expression, andtransduced brain parenchyma cells appeared predominantly inperiventricular regions. This indicates that a fraction of IV deliveredvectors may enter the CNS via the choroid plexus-blood interface. Inadults, substantial staining of brain vasculature was observed. Overalltargeting efficiencies by AAVs to different regions of the brain wasranked as hypothalamus>medulla>cortex>hippocampus>cerebellum>thalamus.EGFP expression was not detected at high levels in neonatal mice thatwere administered rAAV2 or rAAV5 harboring the EGFP reporter gene byinjection of 4×10¹¹ GC per mouse in the superfacial temporal vein. (SeeTable 1 and FIGS. 1A to 1B and 2A to 2B for summary data).

Tissue sections were also immunofluorescently stained with anti-EGFP and-cell type specific marker antibodies to classify EGFP (+) cell types inthe CNS. Detection sensitivity for EGFP (+) cells, particularly neuronsand oligodendrocytes, was improved dramatically. Although differentvectors transduced neurons at variable efficiencies, all 10 vectors(including AAV9) exhibited stronger tropisms to non-neuronal cells,especially astrocytes. One vector (AAV7) targeted oligodendrocytes moreefficiently than the other 9 vectors. Several rAAVs transduced bothneurons and/or astrocytes at higher efficiencies as compared to rAAV9(AAVrh.10, rh.34, and rh.43). Extensive astrocyte transduction wasobserved in hypothalamus and medulla. Injection of certain vectorsresulted in substantial neuron transduction in different regions of thebrain, including neocortex, hippocampus, and hypothalamus. Some vectorsappeared to transduce Purkinje cells in cerebella cortex (e.g., CSp3),while others effectively transduced blood vessel in neocortex, thalamusand hypothalamus. In addition, choroid plexuses in 3^(rd) ventricle,lateral ventricle and 4^(th) ventricle showed strong EGFP expression.EGFP expression was also evaluated in different spinal cord regions ofneonatal and adult mice (results for neonatal studies are shown in FIG.3).

Transduction of non-CNS tissues such as heart and skeletal muscle wasobserved (e.g., for AAV9, AAV8, and CSp3). In some cases, this may leadto some undesirable side effects. To address this issue, miRNA bindingsites were incorporated into the 3′ UTR of the transgene cassette andachieved highly specific and effective detargeting of AAV transductionfrom non-CNS tissues. To inhibit expression in liver, miRNA binding(s)for mR-122 were used. To inhibit expression in skeletal muscle andheart, miRNA binding(s) for mR-1 were used.

TABLE 1 AAV CNS TROPISMS AAV1 AAV2 AAV5 AAV6 AAV6.2 AAV7 AAV8 AAV9 rh.10rh.39 rh.43 CSp3 Adult Cortex + + + ++ ++ +++ ++ + − + Hippocampus + + +++ ++ +++ ++ + − + Thalamus + + + ++ ++ ++++ +++ ++ + + Hypothalamus +++ + +++ ++ ++ +++ +++ + ++ Cerebellum + ++ + ++ +++ +++ ++++ + + +Brain Stem + ++ + ++ ++ ++++ +++ ++ − + Cervical +++ + + +++ +++ +++++++ +++ − + Thoracic +++ + + +++ +++ ++++ +++ ++ − + Lumbar +++ + + ++++++ ++++++ +++ ++ − + Neo-Natal Cortex ++ + − ++ + + +++ ++ ++ ++ ++ ++Hippocampus + + − − − +++ ++ + + ++ ++ + Thalamus + + − − + ++ + + +++ + + Hypothalamus ++ − − + + ++++++ ++++++ + + ++++++ +++ − Cerebellum++ − − + − + + + + ++ + + Brain Stem ++ − − + − ++ + + + +++++ +++ +++Cervical − − − + ++ ++ +++ ++ +++++ ++++ +++ ++ Thoracic + − − + ++ +++++ ++ ++ ++++ +++ + Lumbar + − − + ++ ++ + + ++ +++ +++ + Extent ofTissue Tropsim (− no tropism; ++++++ high tropism) Based on Data inFIGS. 1A to 1B and 2A to 2B.

Example 2: Construction and Evaluation of a Recombinant AAVrh.10 Vectorto Treat CD

Canavan disease (CD) is an inherited neurodegenerative disorder causedby mutations in the aspartoacylase gene (ASPA), leading to accumulationof N-acetyl-aspartic acid (NAA) in oligodendrocytes with resultantspongy degeneration of white matter in the brain. An initial clinicalstudy on rAAV2-based ASPA gene therapy for CD achieved very limitedsuccess. It is believed, without wishing to be bound by theory, that aneffective CD gene therapy will transduce oligodendrocytes throughout theCNS.

A rAAV vector is constructed that comprises a promoter operably linkedwith a region encoding ASPA protein (SEQ ID NO: 13 or 15) as a genetherapy vector for CD. The construct employs CAG (chicken β-actinpromoter with CMV enhancer) to drive the expression of ASPA having acoding sequence as set forth in SEQ ID NO: 14 or 16. The rAAV vector ispackage into rAAV particles using the triple transfection method. Toevaluate its effectiveness, rAAV-ASPA is examined in an ASAP knock-outmouse model of CD for its ability to eliminate or attenuate the CD-likephenotypic of homozygous ASPA knock-out mice (Matalon R et al. TheJournal of Gene Medicine, Volume 2 Issue 3, Pages 165-175). HomozygousASPA knock-out mice exhibit neurological impairment, macrocephaly,generalized white matter disease, deficient ASPA activity and highlevels of NAA in urine. Magnetic resonance imaging (MRI) andspectroscopy (MRS) of the brain of the homozygous mice show white matterchanges characteristic of Canavan disease and elevated NAA levels.Heterozygous ASPA knock-out mice, which have no overt phenotype atbirth, serve as controls.

Example 3: Therapeutic Efficacy and Safety Evaluation of an AAV Vectorto Treat CD

The mouse model of CD is a C57BL/6 derived ASPA gene KO strain. Thehomozygous KO animals present biochemical and neurological defectssimilar to those observed in CD patients. CD mice provide an animalmodel for evaluating gene therapy and other therapeutics for thetreatment of CD. CD mice are used to study the efficacy and safety ofthe novel gene therapy strategies for the treatment of CD.

Experiment Design

To examine therapeutic efficacy and safety, scAAV vectors (e.g., AAV7,AAV8, CSp3 and AAV9) carrying an optimized ASPA expression cassette areinvestigated in a preclinical gene therapy trial of CD. The vectorsinclude miRNA binding site(s) to inhibit ASPA expression in non-CNStissues. Both postnatal day-1 and 3-month-old adult animals are treatedwith each vector at two doses, 1 and 3×10¹⁴ GC/kg by intravenousadministration. For the neonatal CD mice, two litters of animals receiveeach vector at each dose via temporal vein injections for necropsy ofone litter each at 1- and 3-month time points. For the 3-month-old adultCD mice, 12 male animals are treated with each vector at each dose viatail vein injections. Six each of the treated animals are necropsied 1and 3 months later. In further experiments, both postnatal day-1 and3-month-old adult animals are treated with vectors at a dose in a rangeof 10¹¹ to 10¹² GC/subject by direct intraventricular administration.

Functional and Neurological Measurements During the Live Phase of theStudy

1). NAA metabolism. Urine samples are collected from the treated,untreated control, and wild type animals at days 14, 30, 45, 60, 75, and90. The samples are analyzed by HPLC to determine the NAA levels.

2). NAA accumulation and NAA-induced water retention in brain.MRI/MRS-based neuroimaging studies are performed on the live animals inall study groups at 1, 2, and 3 months after the vector treatment tomeasure spectral peak integrals for creatine/phosphocreatine and NAA aswell as abnormal hyperintense areas in the brain.

3). Liver function tests. Serum samples are collected from the animalsin all study groups at days 14, 30, 60, and 90 to measure the levels ofalanine transaminase (ALT) and aspartate aminotransferase (AST) asindicators of vector-related liver toxicity.

4). Neurological tests. Tremors, walking with splayed legs at a slow andshaky pace, and ataxia are among the prominent neurological features ofthe CD mice. At 1, 2, and 3 months after the gene therapy treatment, theanimals in all study groups are subjected to a walking-pattern analysisby staining their feet with color ink and then recording their walkingpatterns as footprints on white paper. The animals also are tested andscored on a rotarod test for their ability to maintain balance.

Enzymatic and Histopathological Analyses at the Endpoints of the Study

1). ASPA activities in the brain and non-CNS tissues. On-target andoff-target expression of ASPA are analyzed by collecting brain, liver,heart and pancreatic tissues at necropsy to measure ASAP activities inthe respective tissue homogenates.

2). Brain white matter and liver pathologies. To examine potentialimprovement in brain white-matter pathology and vector-related livertoxicity resulting from the gene therapy, brain and liver tissues areharvested and fixed, paraffin-embedded and sectioned, and stained withhematoxylin and eosin. Histopathological examination is performed by apathologist.

Example 4: Delivery of Therapeutic Genes to the CNS Cells by AAVrh.10

A screen of different AAV serotypes, was developed to identifycandidates for a therapeutic gene transfer to the CNS. A recombinant AAVvector was constructed that expresses EGFP. The rAAV vector was packagedinto four different AAVs: AAV1, 8, 9 and 10. Adult mice were injectedwith the AAVs into the CSF in the lumbar position. AAV1, 8 and 9transduced cells only in the vicinity of the injection site at thelumber region of the spinal cord following administration of ˜4.8×10¹⁰particles. Surprisingly, AAVrh.10 transduced cells in the gray matteralong the entire spinal cord and brainstem following the same injectionprotocol and dosage as AAV1, AAV8 and AAV9 (FIG. 4A). Recently, AAV9 hasbeen shown to cross the blood brain barrier (BBB) and transduce spinalcord cells after intravenous injection. A weak signal was observed inthe cerebellum and strong signals in the brainstem and spinal cord. Aweak signal (similar to the cerebellum) in the forebrain was alsoobserved. Without wishing to be bound by theory, it is believed that CSFflow and diffusion allows the virus spread along the entire spinal cord,but that the ability of a virus to flow and diffuse depends on thestructure of the viral capsid. The transduced cell types include neuronsand oligodendrocytes. But the majority appears to be astrocytes (FIG.4B), as indicated by overlap of EGFP with GFAP-positive cells.Substantial overlap with the microglia marker, Iba-1 was not observed. Anumber of motor neurons were transduced as indicated by overlap of EGFPexpression and NeuN staining. It was surprising that among theastrocytes, only those situated in the gray matter were transduced andthose that were situated in the white matter and beneath the pia matterwere not transduced. This was striking because the virus is likely to beexposed to astrocytes in these areas since it was administered in thesubarachnoid space.

Example 5: Construction of a Recombinant AAVrh.10 Vector to Treat ALS

An recombinant AAV system was developed as a treatment for ALS. ArAAVrh.10 vector was constructed that expresses a microRNA targetingSOD1 (FIG. 5A). This microRNA was identified as miR-SOD1. The constructemployed CAG (chicken β-actin promoter with CMV enhancer) to drive theexpression of EGFP and miR-SOD1 that was located in an intron in the3′-UTR.

The silencing potency of 9 miRNA constructs was evaluated. Theconstructs were transfected into HEK293 cells. After 48 hours, RNA wasisolated and Northern blot was carried out to detect SOD1 mRNA (FIG.5B). MiR-SOD1#5 (SEQ ID NO: 26) silenced SOD1 expression most potently.Next, miR-SOD1#5 was packaged into AAVrh.10 (FIG. 5D), which was used toinfect HEK293 cells. Total cellular protein was extracted 43 hours afterthe infection and blotted to detect SOD1 (FIG. 5C). Inhibition ofexpression of SOD1 at the protein level was observed.

Example 6: Delivery of Therapeutic Genes to the CNS Cells to Treat ALS

Large batches of AAVrh.10-miR-SOD1 and AAVrh.10-miR-Scr (scrambledmiRNA) were produced using standard techniques. Self-complementary AAV(scAAV) was made because it mediates transduction with higher efficiencythan conventional single stranded AAV [14]. A scAAVrh.10 was tested andfound to express EGFP more rapidly (within 1 week) and stronger than asingle stranded AAV.

AAVrh.10-miR-SOD1 was administered to one group of G93A mice (high SOD1expressers) and AAVrh.10-miR-Scr to another group of G93A mice (n=15).The AAVrh.10 was injected intrathecally into the CSF in the lumbar areaand injected intraventricularly into the forebrain in mice of 60 days ofage (˜4.8×10¹⁰ particles in 8 ul).

The animals were allowed to live their natural lifespan beforesuccumbing to ALS. The lifespan was compared between the two groups. Itwas found that mice receiving the AAVrh.10-miR-SOD1 virus, whichexpresses a SOD1miR5 (SEQ ID NO: 26), lived on average 135 days (±14days), whereas mice receiving the AAVrh.10-miR-Scr, which expresses ascrambled miRNA (SEQ ID NO: 31), lived on average 122 days (±6 days)(FIG. 6B). Moreover, by examining the extent of EGFP expression incervical, thoracic, and lumber spinal cord tissue, a correlation in thelevels of expression in these tissues, particularly with cervicaltissue, and lifespan was observed in AAVrh.10-miR-SOD1 treated mice(FIG. 7A), but not AAVrh.10-miR-Scr treated mice (FIG. 7B). Theseresults suggest that silencing mutant SOD1 expression in the cervicalspinal cord is particularly beneficial in extending survival. A subsetof the animals from each group were perfused with fixative, sectionedand stained for SOD1 in the spinal cord. SOD1 was detected usingstandard techniques [9]. SOD1 staining intensity in EGFP expressingcells was reduced compared with the non-EGFP cells that are transducedwith AAVrh.10-miR-SOD1 (FIG. 6A, showing knockdown of SOD1 expression inastrocytes). Reduction of expression of SOD1 was not observed in cellstransduced with AAVrh.10-miR-Scr.

Tissues from another subset of animals in both groups were dissected toestimate transduction levels. The levels of transduction were estimatedby determining the viral genome content using PCR on DNA samplesobtained from different CNS and non-CNS regions. Measurements in non-CNStissues (e.g. liver) provided an indication of whether virus had leakedto the periphery. Northern and Western analysis was performed to measurethe SOD1 levels in the spinal cord. The antibody used for SOD1 detectionwas polyclonal, sheep anti-human SOD1, by Biodesign International,catalog#K90077C.

Example 7: Combined Intrathecal/Intraventricular Administration Protocol

AAV viruses were injected into mouse CSF by lumbar intrathecal injectionand/or brain third ventricle injection. Injection into mice lumbarsubarachnoid space was carried out using a method modified from Wu etal. [22]. A thin catheter (about 5 cm) was made by stretching PE10 tubeto the inner diameter 0.12 mm. The stretched section was cut to 1.7 to1.9 mm, and two beads (1 mm apart) were made between the thin and thethick sections by heating and pressing the tube. To implant thecatheter, the mouse was anesthetized by injection of Avertin (1.2%2,2,2-tribromoethanol in 2% tert-amyl alcohol and PBS) intraperitoneallyat 0.23 ml/10 g of body weight [23]. The catheter was then implantedbetween the L5 and L6 vertebra. The catheter was stitched to the surfacemuscle at the beaded area. Viruses of dose from 4.80E+10 Genome Copy(for virus screening, in 6 ul) to 2.40E+10 Genome copy (for therapy, in8 ul) were injected via the catheter by a Hamilton syringe at a speed of2 ul/minute. The catheter was sealed at the end by heat and left inplace for one day. Wound was closed by clips. Injection into brain thirdventricle was carried out using a Stoelting Stereotaxic Instrument andmicro-injection pumps from World Precision Instruments followingstandard stereotaxic procedure. Same doses of virus were injected intothe third ventricle at a rate of 1 ul/minutes.

Estimated doses for human and monkeys and comparison with IV injectionare shown below. The two types of monkey are similar in size.

TABLE 2 Estimated Doses for Human and Monkeys Estimated CSF productionEstimate particles/g of Species Avg CSF ml rate ml/hour dose(GC) bodyweight mouse 0.035 0.018 2.40E+10 1.2E+09 human 140 21  9.6E+13 1.3E+09Macaca 14 2.5  9.6E+12 1.7E+09 mulatta (rhesus monkeys) Macaca See,Foust K D, et al., Nature 1.00E+14 2.20E+11  fascicularis Biotechnology,Volume 28, (cynomolgus Number 3, March 2010, 271-274 macaque)

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Example 8. MicroRNA-Regulated, Systemically Delivered rAAV9 Introductionto the Example

This example involves the use of tissue-specific, endogenous microRNAs(miRNAs) to repress rAAV expression outside the CNS, by engineeringperfectly complementary miRNA-binding sites into the rAAV9 genome. Theexample describes recombinant adeno-associated viruses (rAAVs) that cancross the blood-brain-barrier and achieve efficient and stabletransvascular gene transfer to the central nervous system (CNS), whilede-targeting certain other tissues (e.g., liver, heart, skeletal muscleand other tissues) The approaches described in this example allowedsimultaneous multi-tissue regulation and CNS-directed stable transgeneexpression without detectably perturbing the endogenous miRNA pathway.Regulation of rAAV expression by miRNA was primarily via site-specificcleavage of the transgene mRNA, generating specific 5′ and 3′ mRNAfragments.

Gene transfer mediated by recombinant adeno-associated virus (rAAV), asdisclosed herein, is useful for treatment of a large number ofneurological disorders. It has been found that rAAV vectors disclosedherein cross the blood-brain barrier and are specifically expressed inthe CNS. Thus, the vectors may be used for intravascular delivery ofrAAV for gene therapy of CNS diseases, including those that affect largeareas of the brain and spinal cord.

This example describes the use of endogenous microRNAs (miRNAs) tosuppress transgene expression outside the CNS. miRNAs are small,noncoding RNAs that regulate gene expression by post-transcriptionalsilencing. In general, miRNAs may silence genes by two mechanisms. Whenpartially complementary to mRNA sequences, they typically reduce targetmRNA stability and protein expression (e.g., by two- to fourfold orless), a mode of regulation thought to tune mRNA expression. Incontrast, when miRNAs are nearly perfectly complementary to their mRNAtargets, they typically bring about cleavage of the mRNA, triggering itswholesale destruction.

In particular, this example describes the use of miRNAs to detargetrAAV9 expression both separately and concurrently in the liver, heart,and skeletal muscle, the three tissues that are most efficientlytargeted by intravenously delivered rAAV9. Silencing of transgeneexpression in liver, heart, and muscle exploited the natural expressionof the abundant (≥60,000 copies/cell) miRNAs, miR-122, which isexpressed in hepatocytes, and miR-1, a miRNA found in the heart andskeletal muscle of virtually all animals. miR-122-binding sites havebeen successfully used to prevent hepatotoxicity of a transgene from anadenovirus vector. Perfectly complementary sites for miR-1, miR-122, orboth were engineered into the 3′ untranslated region (UTR) of anuclear-targeted, β-galactosidase (nLacZ) reporter transgene whoseexpression was driven by a cytomegalovirus-enhancer, chicken β-actin(CB) promoter. This example presents multiple independent resultsindicating that the miRNAs repress nLacZ expression by cleaving thetransgene mRNA at exactly the same site as by all Argonaute-bound smallRNAs in eukaryotic cells. When delivered systemically in vivo, themiRNA-detargeted rAAV9 vector successfully expressed the reportertransgene in the CNS, but not the liver or heart or skeletal muscle.

Results

miRNAs Efficiently Repress Reporter Gene Expression in Cultured Cells

To evaluate a strategy for rAAV-mediated transduction, one or threetandem copies of a perfectly complementary binding site for miR-1 ormiR-122 were introduced into the 3′ UTR of nLacZ in a rAAV plasmidvector. The constructs were transfected into HuH7 cells, a humanhepatoma cell line expressing—16,000 copies of miR-122 per cell, andmeasured the number of nLacZ-positive cells. The number ofnLacZ-expressing HuH7 cells for the one-site plasmid was about half thatof the no site control; three sites reduced the number ofnLacZ-expressing cells more than sevenfold (FIG. 12A).

Next, expression of the nLacZ constructs was analyzed in human embryonickidney 293 cells, which naturally express low levels of both miR-122 andmiR-1, when miR-1 or miR122 was introduced as a pri-miRNA from a secondplasmid. 293 cells were transfected with the nLacZ reporter plasmidscarrying 0, 1, or 3 miR-122 or miR-1-binding sites, together with aplasmid expressing either pri-miR-122 (FIG. 12B) or pri-miR-1 (FIG.12C). To vary the concentration of the miRNA, either a low (1:3) or ahigh (1:10) molar ratio of the nLacZ-binding site plasmid to the miRNAexpression plasmid was used. When miR-122 or miR-1 was introduced intothe cells, nLacZ expression was repressed only when the nLacZ reportermRNA contained the corresponding miRNA-binding sites; there was noreduction of nLacZ-positive cells when miR-1 was coexpressed with nLacZcontaining miR-122-binding sites or when miR-122 was coexpressed withnLacZ containing miR-1-binding sites (FIGS. 12B and 12C).

Tissue-Specific Endogenous miRNAs Regulate Expression of rAAV9 DeliveredSystemically in Adult Mice

To evaluate miRNA regulation of systemically delivered AAV9CBnLacZvectors in vivo, AAV9CBnLacZ vectors carrying 0, 1, or 3 miRNA-bindingsites perfectly complementary to either miR-122 or miR-1 were produced.The vectors were administered by tail vein injection to adult maleC56BL/6 mice at a dose of 5×10¹³ genome copies per kg (GC/kg) bodyweight. Four weeks later, the liver and heart of the transduced animalswere examined. LacZ staining revealed that the nLacZ transgene wassilenced by the endogenous miRNAs in the cell type and organ in whichthey are predominantly expressed: the transgene was specificallysilenced by miR-122 in the liver and by miR-1 in the heart (FIGS. 13Aand 13B). While nLacZ positive cells were reduced in the livers of theanimals treated with rAAV9CBnLacZ bearing one or three miR-122-bindingsites, nLacZ expression levels in the hearts of the same animals weresimilar to those in the animals treated with AAV9CBnLacZ bearing nosites (FIG. 13A). Similarly, nLacZ expression was not detected in thehearts of the animals that received AAV9CBnLacZ containing one or threemiR-1-binding sites, but nLacZ expression in the livers of the sameanimals was not affected as compared to that in the control animal (FIG.13B). These data suggest that the greater the number of sites for amiRNA in rAAV, the lower the nLacZ expression in the tissue where thecorresponding miRNA was expressed (FIGS. 13A and 13B).

Next, to evaluate whether transgene silencing could be achievedsimultaneously in multiple tissues, different numbers of both miR-122-and miR-1-binding sites were inserted in the 3′ UTR of the rAAV9CBnLacZgenome and examined for their expression in rAAV9 transduced mice.Histochemical staining of tissue sections showed that nLacZ expressionwas suppressed in both heart and liver for rAAV9CBnLac containing one orthree copies each of the miR-1- and miR-122-binding sites, but nLacZ wasreadily detectable in pancreas, where expression of both miR-122 andmiR-1 was low (FIG. 13C). Quantitative, β-galactosidase assays ofhomogenized liver tissue similarly showed that nLacZ expression wassignificantly lower when the transgene contained the miRNA-binding sites(one miR-122-binding site: 7.8±7.4%, P value=0.005; threemiR-122-binding sites: 1.6±1.0%, P value=0.005; one miR-1-plus onemiR-122-binding site: 8.6±5.7%, P value=0.005; three miR-1-plus threemiR-122-binding sites: 3.1±1.2%, P value=0.005; three miR-1-bindingsites: 105.7±11.6%) (FIG. 13D).

miRNA Repression of rAAV Expression does not Perturb Endogenous miRNAPathways

Highly expressed transgenes bearing miRNA-complementary sites have beenreported to promote degradation of the corresponding miRNA. The levelsof miR-122, miR-22, miR-26a, and let-7 were determined in rAAVtransduced liver. No difference in abundance of the four miRNAs wasdetected among the three study groups (FIG. 14A). Moreover, data fromhigh throughput sequencing analyses of small RNA from the livers of oneanimal each from the three study groups show no change in miRNA levels.

In order to determine whether the miRNA-binding sites in the transgenetranscripts would deregulate the expression of the known endogenoustarget mRNAs of miR-122 or miR-1, the expression of cyclin G1, a miR-122target in liver (FIGS. 14B and 14C) and calmodulin, a miR-1 target inheart (FIG. 14D) were analyzed. No significant alteration in cyclin G1or calmodulin expression was detected. miR-122 regulates cholesterolbiosynthesis in the liver, and agents that block miR-122 function mayproduce readily detectable changes in serum cholesterol levels. Nochange in total cholesterol, high-density lipoprotein, or low-densitylipoprotein levels was detected in mice 4 weeks after transduction witheither control rAAV9 or rAAV9 expressing a transgene bearingmiR-122-binding sites (FIG. 14E). It was concluded that in this examplemiRNA-mediated detargeting of rAAV expression had no detectable effecton endogenous miRNA expression or function.

Endogenous miRNAs Silence rAAV Transduction by Site-Specific Cleavage ofTransgene mRNA

To determine how miRNAs suppress expression of transgenes delivered byrAAV in vivo, the transgene mRNA in liver was characterized byconventional PCR (FIG. 15B), quantitative reverse transcription PCR(qRT-PCR) (FIG. 15C), Northern hybridization (FIGS. 15D and 15E), andrapid amplification of 5′ complimentary DNA (cDNA) ends (5′ RACE; FIG.15F). When primers were used that amplify the region between the 3′ endof nLacZ (A⁺F primer) and the 5′ end of the poly(A) signal (APR primer),an amplicon that spans the miRNA-biding sites, a 145 basepair (bp)product was detected after 26 cycles of amplification for the samplesthat received control rAAV. An additional six cycles of amplificationwere required to detect a weak 220 bp band for the samples transduced byrAAV containing three miR-122-binding sites. These data are consistentwith low levels of intact nLacZ mRNA (FIGS. 15A and 15B).

To quantitatively assess the extent of the miRNA-directed repression ofthe transgene transcripts, qRT-PCR was performed using either oligo(dT)or random hexamer primers for reverse-transcription and PCR primer pairsthat span either a 5′ (nLacZ5′F/5′R), or 3′ (nLacZ 3′F13′R) region ofthe nLacZ coding sequence (FIG. 15A). The levels of nLacZ mRNA wereexamined with intact 5′ and 3′ ends in total liver RNA extracted fromfour animals that received the control rAAV9CBnLacZ and four thatreceived rAAV9CBnLacZ containing three miR-122-binding sites in the 3′UTR. Reductions ranging from 3±1 (random hexamer) to 7±1(oligo[dT])-fold in nLacZ mRNA with an intact 3′ end were observed inthe animals that had received rAAV9 containing miR-122-binding sites,relative to the control. In contrast, little or no decrease in nLacZmRNA with an intact 5′ end were detected for the same samples using the5′F/5′R primer pair (FIG. 15C). These results indicate that the primarymode of turnover of the mRNA that has been cleaved by a miRNA was3′-to-5′ exonucleolytic degradation.

To further characterize the fate of the transgene mRNA targeted by miR-1or miR-122, Northern blot analyses was performed. A transgene probebinding to the 5′ end of nLacZ mRNA detected a ˜3.4 kb RNA in an animalinjected with control rAAV9CbnLacZ, the expected size of the of thefull-length nLacZ transcript; a slightly larger band was detected in theliver sample from a mouse treated with rAAV9CBnLacZ bearing threemiR1-binding sites (FIGS. 15A and 15D). In contrast to the singletranscript detected for the rAAV9 expressing nLacZ bearing threemiR-1-binding sites, two RNAs of different sizes were detected for therAAV expressing nLacZ bearing three miR-122 sites (FIG. 15D).

The lengths of these transcripts indicate that the longer transcriptlikely represents the full-length mRNA, whereas the shorter, moreabundant transcript corresponds to 5′ fragments of nLacZ RNA cleaved bymiR-122 at the corresponding miR-122-binding sites in the 3′ UTR (FIG.15D).

To confirm this observation, the Northern analysis was repeated using anRNA probe spanning a portion of 3′ UTR of the transgene mRNA. Inaddition to detecting full-length nLacZ transcripts in the samplestransduced by rAAV9 lacking miRNA-binding sites, two closely migratingspecies smaller than the 281 nucleotide RNA marker were detected. Thesize of these fragments was consistent with miRNA-directed 3′ cleavageproducts of the nLacZ mRNA (FIG. 15E). These two 3′ cleavage productswere also detected by gel electrophoresis of the product from the 5′RACE experiment described below (FIG. 15F).

To determine whether such target cleavage occurs in vivo when the nLacZtranscript contained miR-1 or miR-122-binding sites, rapid amplificationof 5′ cDNA ends (5′ RACE) was performed. FIGS. 16A to 16B present thesequences of 21 clones recovered using 5′ RACE from liver RNA (FIG. 16A)and 22 clones isolated from heart RNA (FIG. 16B) from the animalsinjected with rAAV9 in which the nLacZ 3′ UTR contained three miR-1 andthree miR-122-binding sites. In liver, the sequence signatures formiR-122-directed cleavage of the transgene mRNA were detected at eachmiR-122-binding site: 5% for the first binding site, 48% for the secondbinding site, and 43% for the third binding site. All 5′ ends mapped tothe phosphate that lies between the target nucleotides that pair withpositions 10 and 11 of the sequence perfectly complementary to miR-122,the precise site cleaved by small RNAs bound to Argonaute proteins inall eukaryotes (FIG. 17A). Similar results were obtained in the heartfor the rniR-1 sites (FIG. 17B).

Table 3 presents an expanded 5′ RACE analysis for additional vectorgroups. It was noted that none of the 5′ RACE products sequencedcorresponded to miR-1-directed site-specific cleavage in liver ormiR-122-directed site-specific cleavage in heart (Table 3). Although nocleavage was detected within miR-1-binding sites in the liver, someclones from heart were cleaved within the miR-122-binding sites, but notat the hallmark position for miRNA-directed cleavage.

Intravascularly Delivered rAAV9 can be Efficiently Controlled byEndogenous miRNAs

MiRNA-1 and miRNA-122-binding sites were added into the scAAV9CBenhanced GFP (EGFP) vector genome and injected 10-week-old C57BL/6 malemice with 2×10¹⁴ GC/kg. After 3 weeks, 40 μm sections of brain andspinal cord and 8 μm sections of liver, heart, and skeletal muscle wereprepared and examined for EGFP protein expression. It was found thatintravenously delivered scAAV9CBEGFP efficiently transduced the CNS;EGFP was readily detectable in the thalamus region of the brain and thecervical region of the spinal cord, but also in non-CNS tissues such asliver, heart, and muscle (FIG. 17A). In contrast, transgene expressionin those non-CNS tissues was reduced when miR-1 and miR122-binding siteswere included in the transgene; EGFP expression was unaltered in theCNS, where miR-1 and miR-122 were not present (FIG. 17A). QuantitativeRT-PCR was used to measure the differential expression of therniRNA-repressed EGFP transgene in brain (41.2±7.7%), liver (3.0±0.5%),heart (0.4±0.1%), and muscle (1.3±0.4%), relative to the EGFP transgenelacking miRNA-binding sites (FIG. 17B). To eliminate changes associatedwith transduction efficiency between experiments, the data werenormalized to the number of vector genomes detected in the experimentaland control samples. Similar to the microscopic analyses of native EGFPexpression, the qRT-PCR data show that the presence of miR-122- ormiR-1-binding sites reduced transgene expression in liver (20-fold),heart (100-fold), and muscle (50-fold), but did not detectably altertransgene expression in brain.

Discussion of Results

This example shows that rAAV9 can be engineered so that endogenousmiRNAs repress transgene expression outside the CNS. The resultsindicate that such engineered rAAV9s may be used in therapies for thedegenerating neurons associated with Parkinson's disease, Alzheimer'sdisease and amyotrophic lateral sclerosis, by expressing neurotrophicgrowth factors such as insulin-like growth factor, brain-derivedneurotrophic factor or glial-derived neurotrophic factor in thetransduced astrocytes. This approaches eliminates or lessens non-CNSexpression derived from the peripheral tissues transduced bysystemically delivered rAAV9.

Achieving transgene expression in primarily only the target tissues is aconsideration for the clinical development of safe CNS gene delivery.The results in this example indicate that endogenous miRNAs can beharnessed to restrict the tissue- and cell-type specificity of rAAVexpression, as was initially shown for lentiviral vectors. The datademonstrate that endogenous miRNAs can effectively repress transgeneexpression from rAAV. In both heart and liver, the miRNAs repressedtransgene expression by directing endonucleolytic cleavage of thetransgene mRNA (FIG. 18). MiRNA regulation of rAAV expression did notperturb the expression or function of the corresponding endogenousmiRNA, allowing transgene expression to be restricted to the CNS inmice. The example indicates that a strategy that combines multiplebinding sites for miRNAs expressed in the periphery but not the CNS isuseful for the development of safer, CNS-specific gene therapy vectors.

Materials and Methods

Vector design, construction, and production. Perfectly complementarymiRNA-binding sites were designed based on the annotated miR-1 andmiR-122 sequences in miRBase and inserted into the BstBI restrictionsite in the 3′ UTR of the nLacZ expression cassette of the ubiquitouslyexpressed pAAVCB nuclear-targeted β-galactosidase (nLacZ) plasmid usingsynthetic oligonucleotides (FIG. 15A and Table 3). This vector uses ahybrid cytomegalovirus enhancer/CB promoter cassette that is active inmost cells and tissues. To express miR-122 and miR-1, pri-miR-122 andpri-miR-1 fragments were amplified by PCR from C57/B6 mouse genomic DNA(Table 4) and inserted into the XbaI restriction site 3′ to a fireflyluciferase cDNA in the pAAVCBELuc plasmid. The identity of eachpri-miRNA was verified by sequencing. AAV9 vectors used in this studywere generated, purified, and tittered.

Cell culture and transfection. HEK-293 and HuH7 cells were cultured inDulbecco's modified Eagle's medium supplemented with 10% fetal bovineserum and 100 mg/l of penicillin-streptomycin (Hyclone, South Logan,Utah). Cells were maintained in a humidified incubator at 37° C. and 5%CO₂. Plasmids were transiently transfected using Lipofectamine 2000(Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions.

Mouse studies. Male C57BL/6 mice (Charles River Laboratories,Wilmington, Mass.) were obtained and maintained. To monitor lipidprofiles of the study animals, serum samples were collected 4 weeksafter rAAV9 injection and analyzed for total cholesterol, high-densitylipoprotein and low-density lipoprotein on a COBAS C 111 analyzer (RocheDiagnostics, Lewes, UK). To evaluate endogenous miRNA-mediated,CNS-restricted EGFP gene transfer, 10-week-old male C57BL/6 mice wereinjected intravenously (tail vein) with AAV9CBnLacZ-[miR-122-bindingsite (BS)₁]. AAV9CBnLacZ-(miR-122BS)₃. AAV9CBnLacZ-(miR-1BS)₁.AAV9CBnLacZ-(miR-1BS)₃. AAV9CBnLacZ-(miR-1BS)₁-(miR-122BS)₁, andAAV9CBnLacZ-(miR-1BS)₃-(miR-122BS)₃, respectively, at 5×10¹³ GC/kg bodyweight) or scAAV9CBEGFP at 2×10¹⁴ GC/kg body weight). Animals receivingnLacZ vectors were necropsied 4 weeks later; 8 μm cryosections of liver,heart, and pancreas tissues were prepared for X-gal-histochemicalstaining. Animals that received EGFP vectors were necropsied 3 weekslater and fixed by transcardial perfusion with 4% (wt/vol)paraformaldehyde. Brain, spinal cord, liver, heart, and muscle wereharvested for cryosectioning. Brain and cervical spinal cord tissue werestained as floating sections in a 12-well plate using rabbit anti-EGFPantibody (Invitrogen) diluted 1:500, followed by goat anti-rabbitsecondary antibody (Invitrogen) diluted 1:400. Outside the CNS, EGFPexpression was detected directly by fluorescence. EGFP and antibodyfluorescence was recorded using a Nikon TE-20005 inverted microscope at×10 magnification and an exposure time of 3 seconds for liver, heart,and muscle, and 5 seconds for thalamus (brain) and cervical spinal cord.

Vector genome quantification by qPCR. Genome DNA was extracted from theselected tissues using QIAamp DNA Mini Kit (Qiagen, West Sussex, UK),according to the manufacturer's instructions. Quantitative PCR werecarried out in triplicate using Ring DNA and 0.3 μmol/l EGFP-specificprimers (EGFP-F and EGFP-R) using GoTaq qPCR master mix (Promega,Madison, Wis.) in a StepOne Plus real-time PCR instrument (AppliedBiosystems, Foster City, Calif.).

qRT-PCR analysis. RNA was extracted using Trizol (Invitrogen), accordingto the manufacturer's instructions. Total RNA (0.5-1.0 μg) was primedwith random hexamers or oligo(dT) and reverse-transcribed withMultiScribe Reverse Transcriptase (Applied Biosystems). Quantitative PCRwere performed in triplicate with 0.3 μmol/1 gene-specific primer pairs(nLacZ5′F/5′R, nLacZ 3′F/3′R, cyclinG1F/R and EGFP-F/EGFP-R) using theGoTaq qPCR master mix in a StepOne Plus Real-time PCR device. Thespecificity of qRT-PCR products derived from the 5′ and 3′ ends of nlacZmRNA was confirmed by gel electrophoresis.

Northern blot analysis. Total RNA was extracted from mouse liver andanalyzed by Northern hybridization. To detect nLacZ mRNA, a 618 bpfragment of nLacZ cDNA was isolated by NcoI and PciI digestion ofpAAVCBnLacZ and labeled with α-³²P dCTP by random priming (Takara,Shiga, Japan). To detect 3′ fragments of the cleaved nLacZ mRNA, an 111bp fragment of the poly(A) sequence in the vector genome was cloned intopCR4-TOPO (Invitrogen) for preparation of antisense RNA probe labeledwith α-³²P CTP during in vitro transcription using the Riboprobe SystemT7 kit (Promega). To detect miR-122, miR-26a, miR-22, and let-7 or U6 intotal liver RNA, small RNAs were resolved by denaturing 15%polyacrylamide gels, transferred to Hybond N+ membrane (AmershamBioSciences, Pittsburgh, Pa.), and crosslinked with 254 nm light(Stratagene, La Jolla, Calif.). Synthetic oligonucleotides, 5′end-labeled with γ-³²P ATP using T4 polynucleotide kinase (New EnglandBiolabs, Beverly, Mass.), were used as DNA probes (Table 4) andhybridized in Church buffer (0.5 mol/1 NaHPO₄, pH 7.2, 1 mmol/1 EDTA, 7%(w/v) sodium dodecyl sulphate) at 37° C. Membranes were washed using1×SSC (150 mM sodium chloride, 15 mM sodium citrate), 0.1% sodiumdodecyl sulphate buffer, and then visualized using an FLA-5100 Imager(Fujifilm, Tokyo, Japan).

Western blot analysis. Proteins were extracted withradioimmunoprecipitation assay buffer [25 mmol/1 Tris-HCl, pH 7.6, 150mmol/1 NaCl, 1% (vol/vol) NP-40, 1% (wt/vol) sodium deoxycholate, 0.1%(w/v) sodium dodecyl sulphate] containing a protease inhibitor mixture(Boston BP, Boston, Mass.). Protein concentration was determined usingthe Bradford method (Bio-Rad, Melville, N.Y.). Protein samples, 50 ugeach, were loaded onto 12% polyacrylamide gels, electrophoresed, andtransferred to nitrocellulose membrane (Amersham BioSciences). Briefly,membranes were blocked with blocking buffer (LI-COR Biosciences,Lincoln, Nebr.) at room temperature for 2 hours, followed by incubationwith either anti-GAPDH (Millipore, Billerica, Mass.), anti-cyclin GI(Santa Cruz Biotechnology, Santa Cruz, Calif.) or anti-calmodulin(Millipore) for 2 hours at room temperature. After three washes with PBScontaining 0.1% (vol/vol) Tween-20, membranes were incubated withsecondary antibodies conjugated to LI-COR IRDye for 1 hour at roomtemperature, and then antibodies detected using the Odyssey Imager(LI-COR).

β-Galactosidase assay. Proteins were extracted withradioimmunoprecipitation assay buffer and quantified as described above.Fifty micrograms of protein was used for each β-galactosidase assayusing the Galacto-Star System (Applied Biosystems), according to themanufacturer's instructions.

5′ RACE. 5′ RACE was performed as described. The 5′ RACE Outer Primerand the nLacZ gene-specific primer bGHpolyAR (Table 4) were used for thefirst round of nested PCR. The 5′ RACE Inner Primer and the nLacZgene-specific primer nLacZpolyR, which is located near the stop codon ofnLacZ cDNA, were used for the second round of nested PCR (Table 4). PCRproducts were TOPO-cloned into pCR-4.0 (Invitrogen) and sequenced.

Statistical analysis. All results are reported as mean±SD and comparedbetween groups using the two-tailed Student's t-test.

TABLE 3 Summary of microRNA-guided transgene mRNA cleavage in mouseliver and heart Cleavage site Between Between Between Random miR BScleavage Position 10 and 11 nt 17 and 18 nt 18 and 19 nt site Liver 1Copy of miR-122 BS (21 clones) 1 17/21  81% ND ND 19%  3 Copies ofmiR-122 BS (11 clones) 1 ND 100% ND ND 0% 2  4/11 3  7/11 3 Copies eachof miR-1 and miR 122 1 ND ND ND ND 0% miR 1 3 × BS BS in a single vector(21 clones) 2 ND 3 ND miR-122 3 × BS 1  1/21  95% ND ND 5% 2 10/21 3 9/21 Heart 1 Copy of miR-1BS (12 clones) 1 12/12 100% ND ND 0% 3 Copiesof miR 1BS (21 clones) 1 ND  80% 4/21 20% ND 0% 2 16/21 ND 3  1/21 ND 3Copies each of miR 1 and miR 122 1 ND ND ND 1/22 14% 4% miR-122 3 × BSBS in a single vector (22 clones) 2 ND 1/22 3 ND ND miR 1 3 × BS 1  1/22 73% ND  9% ND 0% 2  7/22 1/22 3  8/22 1/22

TABLE 4 Oligonucleotide primers and probes used in Example 8.Oligo nucleotides Sequence SEQ ID NO (miR-1)₁ sense[PHOS]CGAAATACATACTTCTTTACATTCC SEQ ID NO: 32 ATT (miR-1)₁ anti-sense[PHOS]CGAATGGAATGTAAAGAAGTATGT SEQ ID NO: 33 ATTT (miR-122)₁ sense[PHOS]CGAAACAAACACCATTGTCACACT SEQ ID NO: 34 CCATT (miR-122)₁ anti-sense[PHOS]CGAATGGAGTGTGACAATGGTGTTT SEQ ID NO: 35 GTTT (miR-1)₃ sense[PHOS]CGAAATACATACTTCTTTACATTCC SEQ ID NO: 36AATACATACTTCTTTACATTCCAATACATA CTTCTTTACATTCCATT (miR-1)₃ anti-sense[PHOS]CGAATGGAATGTAAAGAAGTATGT SEQ ID NO: 37ATTGGAATGTAAAGAAGTATGTATTGGAA TGTAAAGAAGTATGTATTT (miR-122)₃ sense[PHOS]CGAAACAAACACCATTGTCACACT SEQ ID NO: 38CCAACAAACACCATTGTCACACTCCAACA AACACCATTGTCACACTCCATT(miR-122)₃ anti-sense [PHOS]CGAATGGAGTGTGACAATGGTGTTT SEQ ID NO: 39GTTGGAGTGTGACAATGGTGTTTGTTGGA GTGTGACAATGGTGTTTGTTT (miR-1)₁-(miR-122)₁[PHOS]CGAAATACATACTTCTTTACATTCC SEQ ID NO: 40 senseAACAAACACCATTGTCACACTCCATT (miR-1)₁-(miR-122)₁[PHOS]CGAATGGAGTGTGACAATGGTGTTT SEQ ID NO: 41 anti-senseGTTGGAATGTAAAGAAGTATGTATTT Synthesized (miR-1)₃-TTCGAACTCGAGATACATACTTCTTTACAT SEQ ID NO: 42 (miR-122)₃ fragmentTCCAATACATACTTCTTTACATTCCAATAC ATACTTCTTTACATTCCACCATGGACTAGTACAAACACCATTGTCACACTCCAACAAAC ACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAGCGGCCGCTTCGAA Pri-miR-122F ATCGGGCCCGACTGCAGTTTCAGCGTTTGSEQ ID NO: 43 Pri-miR-122R CGCGGGCCCGACTTTACATTACACACAAT SEQ ID NO: 44Pri-miR-1F CGCGGGCCCGACTGATGTGTGAGAGAGAC SEQ ID NO: 45 Pri-miR-1RCGCGGGCCCGACTTTCGGCCTCCCGAGGC SEQ ID NO: 46 nLacZ5¢F(5¢F)TGAAGCTGAAGCCTGTGATG SEQ ID NO: 47 nLacZ¢5R(5¢R) GAGCACCTGACAGCATTGAASEQ ID NO: 48 nLacZ3¢F(3¢F) CTCAGCAACAGCTCATGGAA SEQ ID NO: 49nLacZ3¢R(3¢R) TTACTTCTGGCACCACACCA SEQ ID NO: 50 nLacZpolyF(A⁺F)TGGTGTGGTGCCAGAAGTAA SEQ ID NO: 51 nLacZpolyR(A⁺R) CAACAGATGGCTGGCAACTASEQ ID NO: 52 bGHpolyAR(bGH⁺PAR) TGGGAGTGGCACCTTCCA SEQ ID NO: 53 EGFP-FCGACCACTACCAGCAGAACA SEQ ID NO: 54 EGFP-R CTTGTACAGCTCGTCCATGCSEQ ID NO: 55 CyclinG1F AATGGCCTCAGAATGACTGC SEQ ID NO: 56 CyclinG1RAGTCGCTTTCACAGCCAAAT SEQ ID NO: 57 MM-ActinF ATGCCA ACACAGTGCTGTCTGGSEQ ID NO: 58 MM-ActinR TGCTTGCTGATCCACATCTGCT SEQ ID NO: 59miR-122 probe TGGAGTGTGACAATGGTGTTTG SEQ ID NO: 60 Let-7 probeAACTATACAACCTACTACCTCA SEQ ID NO: 61 miR-26a probeAGCCTATCCTGGATTACTTGAA SEQ ID NO: 62 miR-22 ProbeACA GTT CTT CAA CTG GCA GCT T SEQ ID NO: 63 U6 probeCTCTGTATCGTTCCAATTTTAGTATA SEQ ID NO: 64

Example 9: Intravenous Injection of rAAVs Mediated WidespreadTransduction in Neonatal Mouse CNS Introduction to the Example

This example describes an analysis of nine scAAV vectors for CNS genetransfer properties after systemic administration. This study involvedidentifying more effective vectors for the CNS gene transfer, In someaspects the study examined serotypes or natural variants of rAAVs forenhanced-permeation of the BBB. In some cases, the study sought toidentify rAAV vectors with improved delivery of enhanced greenfluorescent protein (EGFP) to the CNS following facial vein injection onpostnatal day 1 (P1). AAV9 was included in the study. Except for rAAV2and rAAV5, all other 7 vectors crossed the BBB with varied transductionefficiency, among which rAAVrh.10, rAAVrh.39, rAAVrh.43, rAAV9 andrhAAV7 rank in the top 5, mediating robust EGFP expression in bothneuronal and glial cells throughout the CNS in this study. Theperformance of rAAVrh.10 was comparable to that of rAAV9 and in somecase better. Several rAAVs efficiently transduce neurons, motor neurons,astrocytes and Purkinje cells; among them, rAAVrh.10 is at least asefficient as rAAV9 in many of the regions examined. Intravenouslydelivered rAAVs did not cause abnormal microgliosis in the CNS. TherAAVs that achieve stable widespread gene transfer in the CNS are usefulas therapeutic vectors for neurological disorders affecting largeregions of the CNS as well as convenient biological tools forneuroscience research.

Results

Twenty one days after vector administration in P1 mice, the CNStransduction profiles of the following recombinant AAV vectors encodingEGFP: rAAV1, rAAV2, rAAV5, rAAV6, rAAV6.2, rAAV7, rAAV9, rAAVrh.10,rAAVrh.39 and rhAAVrh.43 were compared. The vectors used in this studywere comparable in purity and morphological integrity (FIGS. 19A to19D). As assessed by the scoring system described in the methods, rAAV9was among the top performers; most other rAAVs tested (rAAV1, rAAV6,rAAV6.2, rAAV7, rAAVrh.10, rAAVrh.39 and rAAVrh.43) also gave rise toEGFP expression throughout the CNS (Table 2). The number of apparentEGFP positive cells (Table 5) among sub-anatomical structures wasinfluenced by the particular vector used. For these seven rAAVs, andrAAV9 (total of eight rAAVs), that permeated the BBB and accomplishedCNS transduction after i.v. delivery, EGFP positive cells were found inhypothalamus followed by medulla, striatum, hippocampus, cortex andcerebellum. In contrast, the transduction efficiency in olfactory bulband thalamus was relatively low (Table 5). A quantitative assessment ofEGFP gene transfer efficiency was made of each rAAV. 12 sub-anatomicallyand functionally important regions in the brain were selected forquantitative analysis of the mean EGFP intensity/pixel in each regionfor each rAAV by using Nikon NIS elements AR software V. 32 (FIGS. 19Ato 19C) (see Methods). For the eight vectors that achieved CNStransduction after i.v. injection, the mean EGFP intensity/pixel wasrelatively low in cortex, habenular nucleus, cornu ammonis, dentategyrus, thalamus, cerebellum and olfactory bulb, moderate in choroidplexus and caudate-putamen, but high in hypothalamus, medulla andamygdale (FIGS. 19A to 19C). The average EGFP intensities of all 12regions for different rAAVs were compared in FIG. 19D. AAVrh.10,AAVrh.39 and AAVrh.43 were noted for gene transduction efficiency inbrain, followed by AAV7, AAV9, and AAV1 (FIGS. 19A to 19D). Those eighteffective serotypes also mediated EGFP expression throughout the spinalcord, to different degrees. The same quantitative analysis was performedfor each rAAV in the cervical, thoracic and lumbar sections of thespinal cord (FIGS. 19A to 19C); the average EGFP intensities of thethree sections for different rAAVs were also compared (FIG. 19D). AAV1,AAV9, AAVrh10, AAV.rh39 and AAV.rh43 displayed strong transduction inthe spinal cord with the high EGFP intensity observed in the cervix,followed by thoracic and lumbar sections of the spinal cord (FIGS. 19Ato 19D). For rAAV2 there were few EGFP-positive cells in hippocampus,cortex and hypothalamus. EGFP-positive cells were observed in thehypothalamus in AAV5-injected mice. A description of the observationsmade in different CNS structures is provided below. The subanatomic CNSstructures may serve as a target for CNS gene therapy. In some cases,the subanatomic CNS structures are associated with pathological changesin one or more neurological disorders. In some cases, the subanatomicCNS structure have distinct transduction profiles for one or more rAAVs.

Striatum. Pathology of the striatum is associated with Huntington'sdisease, choreas, choreoathetosis, and dyskinesias. Addiction mayinvolve plasticity at striatal synapses. Systemic injection of rAAV9 inneonatal mice tranduces striatal tissue. In this study, a large numberof cells with neuronal morphology in this region were also transduced byrAAVrh.10 (FIG. 20), which was confirmed by co-staining with a neuronalmarker as described below. Other vectors, including rAAVrh.39 and rAAV7,also mediated moderate transduction in striatum (FIG. 20). In contrast,rAAV6, rAAV6.2, and rAAV1 resulted in relatively lower EGFP expressionin this structure (FIG. 20).

Hippocampus. The hippocampus is a region associated with long-termmemory and spatial navigation, which is usually damaged by stress andpathogenesis of diseases such as epilepsy and Schizophrenia. Largenumbers of EGFP-positive neurons were observed bilaterally in allregions of the hippocampus, namely dentate gyrus, hilus, CA1, CA2 andCA3 for the mice received intravenous rAAVrh.10, rAAV9, rAAV7,rAAVrh.39, and rAAVrh.43 (ranked by transduction efficiency in thisstructure, Table 5 and FIGS. 19A-19D and FIG. 20). In addition to theneuronal transduction pattern, EGFP-positive cells had morphologicappearance of astrocytes (FIG. 20). This was further confirmed by doublestaining with antibodies against EGFP and astrocytic marker as describedbelow. For intravenously delivered rAAV1, rAAV6 and rAAV6.2 vectorsthere were small numbers of EGFP-positive cells in the hippocampus (FIG.20).

Cortex. Pathological changes in the cortex have been implicated inAlzheimer's and Parkinson's diseases. AAV7, AAV9, AAVrh.10, AAVrh.39 andAAVrh.43 vectors achieved moderate EGFP transduction in cortex (Table 5and FIGS. 19A to 19D and 20). The morphology of transduced cells wasconsistent with both neurons and astrocytes as further confirmed bycellular marker staining and confocal microscopic analysis describedbelow. Prominent EGFP-positive cells were typically observed in theventrolateral regions of the cortex, including posterior agranularinsular cortex, piriform cortex, lateral entorhinal cortex,posterolateral cortical amygdaloid nucleus and posteromedial corticalamygdaloid nucleus (FIG. 20). Strong EGFP signals spread from +1.5 to−3.3 mm in relation to the Bregma (0.0 mm). The cortical transductionefficiency of rAAVrh.10, rAAV9, rAAVrh.39 and rAAVrh.43 was comparable(Table 5 and FIGS. 19A to 19D and 20). AAV1, AAV6 and AAV6.2 vectorsalso transduced cells in the cortex (FIG. 20).

Hypothalamus. A role for the hypothalamus is to secret neurohormones tocontrol certain metabolic processes. The hypothalamus is also indicatedin the etiology of diabetes. EGFP signal was observed in thehypothalamus for eight vectors. Intravenous administration of rAAVrh.10resulted in the highest EGFP expression in the entire hypothalamus,followed by rAAVrh.39, rAAV7, rAAV6.2, rAAVrh.43, rAAV9, rAAV1 and rAAV6(FIGS. 19A to 19D and 20 and Table 5). Interestingly most EGFP-positivecells in this structure have an astrocytic morphology which wasascertained by immunostaining for an astrocytic cell type specificmarker as described below. The astrocytic EGFP signal tended to obscuredirect examination of morphological details of other transduced cells.However, this was clarified by double immunofluorescent staining oftissue sections with antibodies for EGFP and neuronal cell markers asdescribed below.

Cerebellum. The pathological lesions in cerebellum are often found indiseases such as cerebellar-cognitive affective syndrome, developmentalcoordination disorder, posterior fossa syndrome, linguistic deficits,aging, attention deficit hyperreactivity disorder, autism, dementia andschizophrenia. EGFP-positive cells and fibers were detected incerebellum for most rAAV vectors (Table 5 and FIGS. 19A to 19D and 20).A large number of EGFP-expressing cells were found in the Purkinje andgranule cell layers for rAAV7, rAAV9, rAAVrh.10, rAAVrh.39 and rAAVrh.43(FIG. 20). The transduction profile of rAAV1 vector indicated expressionin cells in the granule cell layer, while rAAV6 and rAAV6.2 werelocalized in cells in the Purkinje cell layer (FIG. 20).

Medulla. The medulla is a potential gene therapy target for treatingchronic pain. Most rAAVs mediated moderate to robust EGFP expression inmedulla with most green cells being present in the outer rim (FIG. 20).Transduction efficiencies of these rAAV in this region are ranked in thefollowing order:rAAVrh.39=rAAVrh.43>rAAV.rh10>rAAV1>rAAV9>rAAV7>rAAV6.2>rAAV6 (Table 5and FIG. 19A). The morphology of most EGFP-transduced cells wasconsistent with the cells being astrocytes.

Spinal cord. The spinal cord is involved with motor neurons diseases.rAAVrh.10, rAAV9, rAAVrh.39 and rAAVrh.43 gave rise to very robust EGFPexpression in cervical gray and white matter, while rAAV1, rAAV6.2 andrAAV7 showed moderate EGFP intensity (Table 5 and FIGS. 19A to 19D and21). For rAAV1 the EGFP signal was observed in white matter. Thetransduction ability of all effective rAAVs decreased from cervical tolumbar spinal cord. EGFP-positive cells were visible in the latterregion. Large populations of EGFP-positive cells with astrocyticmorphology were observed throughout the spinal cord (FIG. 21). Inaddition, rAAVrh.10, rAAV9, rAAVrh.39, rAAVrh.43 and rAAV7 alsotransduced cells with motor neuron morphology in the ventral regions ofspinal cord (FIG. 21). Ascending dorsal column fibers showed clear EGFPsignal. In addition, dorsal root ganglia (DRG) displayed remarkabletransduction with strong EGFP expression in DRG neurons (FIG. 22 andFIG. 26). The identities of rAAV transduced cell types in the spinalcord were characterized by co-immunofluorescence staining withantibodies against EGFP and cell type specific markers as describedbelow.

IV Administration of AAV Vectors Leads to Transduction of Different CellTypes in the CNS

To confirm the identity of transduced cells in different regions of theCNS, double immunofluorescent staining was performed with antibodies forEGFP and NeuN (generic neuronal marker), glial fibrillary acid protein(GFAP; astrocyte marker), calbindin-D28K (Purkinje cell marker), andcholine acetyl transferase (ChAT; motor neuron marker) (FIG. 23). Theimmunostaining results showed that a large number of NeuN positive cellsexpressed EGFP throughout the mouse brain, which indicated widespreadneuronal transduction. The regions with high density of transducedneurons included striatum, hippocampus, cortex and hypothalamus.rAAVrh.10, rAAV9, rAAV7 and rAAVrh.39 vectors were efficient inmediating neuronal transduction, followed by AAV6.2, AAV1 and AAV6(FIGS. 19A to 19D and 23). In addition, dopaminergic neurons insubstantia nigra were transduced by AAV.rh10 (FIG. 23). Transduced cellsin the CNS included GFAP-positive astrocytes with small cell bodies andhighly ramified processes (FIG. 23). The calbindin-D28K immunostainingconfirmed the identity of a number of transduced cells in the cerebellumas Purkinje cells, with EGFP expression in both cell body and theirtree-like processes (FIG. 23). The rAAVs proficient in transducingPurkinje cells include: rAAVrh.10, rAAV9, rAAVrh.39, rAAV7, rAAV6.2 andrAAVrh.43. rAAV1 and rAAV6 transduced a portion of Purkinje cells withrelatively low EGFP intensity (FIGS. 19A to 19D). Transduction of motorneurons was confirmed by the presence of large EGFP+/ChAT+ cells in theventral spinal cord for several rAAV vectors (FIG. 23). rAAVrh.10,rAAV9, rAAV7, rAAVrh.39 showed comparable efficiency transduction ofmotor neurons (FIG. 21).

IV Administration of AAV Vectors Mediated Robust Transduction inVentricles and Brain Blood Vessels

EGFP expression was observed in the choroid plexus cells in lateral,3^(rd) and 4^(th) ventricles of the animals infused with rAAVrh.39,rAAVrh.10, rAAVrh.43, rAAV7 and rAAV9 (ranked by transductionefficiency, Table 5 and FIGS. 19A to 19D and 24). EGFP expression indifferent ventricles of the same mouse brain was similar (FIG. 24).Ependymal cells lining the ventricles were also transduced. Anobservation regarding the distribution of EGFP-positive cells was theapparent gradient with the highest number of transduced cells inperi-ventricular regions and progressively lower numbers with increasingdistance to the ventricles. This was apparent in areas around the 3^(rd)and 4^(th) ventricles than the lateral ventricles (FIG. 24). ExtensiveEGFP signal was also found with blood vessels throughout mouse brain andspinal cord. This was verified by dual immunofluorescent staining withantibodies directed to EGFP and a blood vessel endothelium specificmarker, CD34 (FIGS. 27A and 27B). Unlike the rAAV transduction profilesin different regions of the brain parenchyma, the EGFP transduction ofthe blood vessels throughout the CNS was relatively uniform for anygiven vector. However, transduction of blood vessels was influenced bythe particular rAAV used. A majority of rAAVs mediated moderate (e.g.,rAAV6) to highly efficient (e.g. rAAVrh.10) blood vessel transduction inthe CNS.

IV Injection of AAV Vectors Did not Cause Microgliosis

Brain sections were also stained with antibody against Iba-1 to labelmicrogial cells. The Iba-1-positive cells in the sections from micereceived rAAVrh.10 was no more than those in naïve or PBS-injected mice(FIG. 28). This result indicated that intravascularly delivered rAAVs donot cause sustained inflammation in the CNS of mice 3 weeks after theinjection of P1 neonates.

Discussion of Results

In this study, the CNS transduction profile was evaluated for 10different rAAV vectors delivered by intravascular infusion in neonatalmice. Most of the rAAVs can cross the BBB and mediate gene transfer tothe neonatal mouse CNS with varying degrees of efficiency (FIGS. 19A to19D, 20 and 21 and Table 5). After systemic administration, rAAVrh.10,rAAVrh.39, rAAVrh.43, and rAAV9 are the effective rAAVs with similartransduction capabilities and cellular tropism, as assessed by overallEGFP expression in the CNS. Specifically, a number of regions in themouse CNS, including striatum, hippocampus, cortex, hypothalamus,cerebellum, medulla, and cervical spinal cord, revealed substantial EGFPexpression. In addition, rAAV6.2 and rAAV7 were also effective. AAV1 andAAV6, achieved CNS transduction (Table 5). Native EGFP expression wasdetectable in brain and spinal cord sections for most of the rAAVswithout immunostaining (FIG. 29).

This example has clinical significance for gene therapy of CNS-relateddisorders, including for young patients. For a variety of neurologicaldiseases, early treatment during infancy may be necessary to preventirreversible CNS injury. The capacity of rAAVs to transduce largenumbers of neuronal cells in different regions is relevant for treatingneurological diseases such as spinal muscular atrophies, neuronal ceroidlipofuscinoses, and spinocerebellar degenerations. The efficiency ofsome rAAV vectors in transducing Purkinje and granule layer cellsindicates that the vectors may be used for treating spinocerebellarataxias. Transduction of astrocytes by rAAVs expressing secretedneurotrophic factors may be also beneficial for a number ofneurodegenerative diseases such as Canavan's disease and amyotrophiclateral sclerosis. The vascular transduction in the CNS may be relevantfor treating brain ischemia and stroke. The clinical application ofintravascular rAAV-mediated gene delivery may also extend to theperipheral nervous system (PNS). Efficient transduction of DRG providesnew therapeutic strategies for patients suffering from chronic pain.

Systemic gene delivery to the CNS is also useful as a method tomanipulate gene expression in research. Effective and stable transgeneexpression in the CNS by intravenous administration of rAAVs may beapplied to establish somatic transgenic animal models, which is apotentially cheaper, faster and simpler method than conventionaltransgenesis. Somatic CNS gene knock-down animal models may also becreated using the method described herein.

Some rAAVs indeed demonstrated unique transduction profiles in the CNS.For instance, rAAV1 displayed transduced granule cells in thecerebellum, while rAAV6 and rAAV6.2 transduced Purkinje cells, andothers transduced both types of cells (FIG. 9). This indicates that onceacross the BBB, the rAAVs have distinct tropisms, which can beattributed to the capsid because that the vector genome used in allvectors was the same.

AAV serotypes disclosed herein can efficiently transduce brain capillaryendothelial cells, neurons and astrocytes. This indicates that thesevectors may extravasate from the circulation and reach the CNSparenchyma, possibly by crossing the BBB. AAV may cross the endothelialbarrier by a transcytosis pathway. In this study, choroid plexuses andtheir surrounding parenchymal tissue were efficiently transduced. Inaddition, there was an apparent gradient of EGFP intensity fromperi-ventricular (higher) to deep parenchymal (lower) tissue. Theseobservations indicate that AAV may enter the neonatal mouse CNS throughthe choroid plexus, followed by widespread distribution via CSF and/orinterstitial fluid flow to transduce neuronal and glial cells.

Neuronal- or glial-specific promoters, such as synapsin-1, and GFAPpromoters may be used to restrict gene expression to a specific celltype. A further method to achieve targeted CNS gene delivery is toutilize RNA interference to detarget the peripheral tissues bypost-transcriptional regulatory mechanisms. By adding microRNA bindingsites into the 3′ end of the transgene cassettes, transgene expressionafter systemic administration of AAV vectors may be reduced oreliminated in tissues such as liver, heart and skeletal muscle, whilemaintaining CNS transduction.

Materials and Methods

AAV Production

ScAAV vectors were produced by trans-encapsidation of rAAV vector genomeflanking by inverted terminal repeats (ITRs) from AAV2 with the capsidsof different AAVs using the method transient transfection of 293 cellsand CsCl gradient sedimentation as previously described. Vectorpreparations were titered by quantitative PCR. Purity of vectors wasassessed by 4-12% SDS-acrylamide gel electrophoresis and silver staining(Invitrogen, Carlsbad, Calif.). Morphological integrity of each vectorused in the study was examined by transmission electron microscopy ofnegative stained recombinant AAV virions. The expression of EGFP in thescAAV vector genome is directed by a hybrid CMV enhancer/chicken β-actinpromoter.

Neonatal Mouse Injections

Wild-type C57BL/6 mice littermates were used. Mice breeding wereconducted using programmatic timing method. Pregnant mice were monitoreddaily from embryonic day 17 to 21 to ensure the newborn pups could bedosed with vectors on P1. The mother (singly housed) of each litter tobe injected was removed from the cage. Vectors were diluted toconcentration of 4×10¹² GCs/mL in PBS and 100 μl of solution wassubsequently drawn into 31G insulin syringes (BD Ultra-Fine II U-100Insulin Syringes). P1 pups of C57BL/6 mice were anesthetized usingisoflurane and rested on ice. For intravenous injections, a dissectionmicroscope was used to visualize the temporal vein (located justanterior to the ear). The needle was inserted into the vein and theplunger was manually depressed. Correct injection was verified by notingblanching of the vein. Each pup received 4×10¹¹ GCs of differentscrAAVCBEGFP vectors (rAAV1, rAAV2, rAAV5, rAAV6, rAAV6.2, rAAV7, rAAV9,rAAVrh.10, rAAVrh.39, rAAVrh.43; n=6-8 mice per group) via thesuperficial temporal vein. After the injection pups were carefullycleaned, rubbed with their original bedding, and then returned to theiroriginal cage. The mother was then reintroduced to the cage after briefnose numbing using ethanol pads.

Histological Processing

The study animals were anesthetized 21 days post-injection, thentranscardially perfused with 15 mL of cold PBS followed by 15 mL offixation solution containing 4% paraformaldehyde (v/v) with 0.2% ofglutaraldehyde (v/v) in PBS. Then the whole carcasses were post-fixed infixation solution for 5 days. Spinal cords and brains were extractedunder a bright-field dissecting microscope, rinsed in PBS, and thencryoprotected in 30% sucrose (w/v) in PBS at 4° C. Once the tissues sankto the bottom of the sucrose solution, they were embedded in Tissue-TekOCT compound (Sakura Finetek, Torrance, Calif.) and frozen in a dryice/ethanol bath. The tissue blocks were stored at −80° C. untilsectioning. Serial 40 μm floating sections of the entire brain were cutin a Cryostat (Thermo Microm HM 550). For the spinal cord, 3 mm lengthsections were taken from cervical, thoracic and lumbar regions, and thenserial 40 μm transverse sections prepared as above.

Immunostaining and Microscopy Imaging Analysis

Brain and spinal cord sections were stained as floating sections in12-well plates. Sections were washed 3 times in PBS for 5 min each time,and then incubated in blocking solution containing 1% Triton-X100 (v/v)(Fisher, Pittsburgh, Pa.), 5% dry-milk (w/v) and 10% goat serum (v/v)(Invitrogen) for 2 h at room temperature. Then the sections wereincubated with primary antibodies diluted in blocking solution at 4° C.overnight. The following day tissue sections were washed twice in 0.05%Tween-20 (v/v) in PBS (PBST) and once with PBS, with each washing steplasting 10 min. Afterwards sections were incubated with appropriatesecondary antibodies in blocking solution at room temperature for 2 h.Sections were washed again as above before mounting on glass slides.Vectashield with DAPI (Vector Laboratories, Burlingame, Calif.) was usedto coverslip all slides, and then they were analyzed using a fluorescentinverted microscope (Nikon Eclipse Ti) or a Leica TSC-SP2 AOBS confocalmicroscope equipped with a 63× oil lens and a DM-IRE2 invertedmicroscope. The primary antibodies used in this study were as follows:rabbit anti-GFP (Invitrogen), goat anti-ChAT and mouse anti-NeuN (bothfrom Millipore, Billerica, Mass.), mouse anti-GFAP (Cell signaling,Danvers, Mass.), rat anti-CD34 (Abcam, Cambridge, Mass.), mouseanti-Calbindin D-28k (Sigma, St Louis, Mo.) and rabbit anti-DARPP(Abcam, Cambridge, Mass.). The secondary antibodies used in the studyincluded: DyLight 488 AffiniPure Donkey Anti-rabbit IgG (JacksonImmunoResearch, West Grove, Pa.); DyLight 549 AffiniPure DonkeyAnti-Goat IgG (Jackson ImmunoResearch); DyLight 549 Affinipure GoatAnti-Rat IgG (Jackson ImmunoResearch); DyLight 594 AffiniPure GoatAnti-Mouse IgG (Jackson ImmunoResearch); goat anti-rabbit IgG-Alexafluro 488 (Invitrogen) and goat anti-mouse IgG-Alexa fluro 568(Invitrogen).

Semi-Quantitative and Quantitative Comparison of EGFP Transduction byDifferent Vectors

To generate a quantifiable and comparable data format, asemi-quantitative scoring system was develop to estimate transductionefficiency of different rAAV vectors in different regions of the mouseCNS. Briefly, regions with no EGFP positive cells were marked as (−).Regions with very few EGFP positive cells were scored (+), regions withsome EGFP positive cells were ranked as (++), regions with many EGFPpositive cells were marked as (+++). Finally, regions filled with EGFPpositive cells were marked as (++++).

Next, 12 sub-anatomically and functionally important regions in thebrain as well as cervical, thoracic and lumbar sections of the spinalcord were selected for quantitative analysis of images that were takenon a Nikon Eclipse Ti inverted microscope equipped with a Retiga 2000-RVCCD cooled camera. Nikon NIS elements AR software v. 3.2 was used forintensity quantification. Prior to quantification, optimal light sourceintensity and exposure times were obtained by plotting anintensity/exposure time curve using fluorescence reference slides (TedPella, prod. 2273). It was found that the intensity and exposure timeshad linear correlation. In addition, overexposure and extremeunderexposure distorts the linear correlation. The maximum intensity(ND1) and a 20 ms exposure were used for all sections to avoidoverexposure. For quantification, fixed region of interest (ROI) wasused to quantify the brightest area of any given brain region. A meanintensity (total intensity/size of ROI) was obtained for each region ofall serotypes.

TABLE 5 Transduction characteristics of AAV serotypes followingintravascular injections into neonatal mouse brain score n score n scoren score n score n score n Olfactory Bulb Striatum Hippocampus CortexThalamus Hypothalamus AAV1 + 3 ++ 3 ++ 3 ++ 3 + 3 +++ 3 AAV2 − 3 − 3 +3 + 3 + 3 + 3 AAV5 − 3 − 3 − 3 + 3 − 3 + 3 AAV6 + 3 + 3 ++ 3 ++ 3 + 3+++ 2 ++ 1 AAV6.2 − 3 +++ 2 ++ 3 ++ 3 + 3 ++++ 3 ++ 1 AAV7 +++ 1 +++ 3+++ 2 ++ 3 + 3 ++++ 3 ++ 2 ++ 1 AAV9 +++ 2 +++ 3 ++ 3 +++ 1 + 3 ++++ 1++ 1 ++ 2 +++ 2 rh10 +++ 1 ++++ 1 +++ 3 +++ 2 ++ 2 ++++ 3 ++ 2 ++ 2 ++1 + 1 rh39 +++ 1 ++++ 2 +++ 3 +++ 1 + 3 ++++ 3 ++ 2 +++ 1 ++ 2 rh43 ++ 3+++ 3 +++ 3 +++ 3 + 3 ++++ 1 +++ 2 Choroid Cerebellum Medulla CervicalThoracic Lumber Plexus AAV1 +++ 3 +++ 3 +++ 3 +++ 3 + 3 +++ 3 AAV2 + 3 +3 + 3 − 3 − 3 ++ 3 AAV5 − 3 − 3 − 3 − 3 − 3 − 3 AAV6 ++ 3 ++ 3 ++ 3 +3 + 3 +++ 3 AAV6.2 ++ 3 ++ 3 ++ 3 ++ 3 + 3 ++++ 3 AAV7 +++ 1 ++ 3 ++ 3 +3 + 3 ++++ 3 ++ 2 AAV9 +++ 1 ++ 3 ++++ 1 ++ 3 + 3 ++++ 3 ++ 2 +++ 2 rh10+++ 1 +++ 3 ++++ 1 ++ 3 + 3 ++++ 3 ++ 2 +++ 2 rh39 +++ 1 ++++ 1 ++++ 1+++ 3 + 3 ++++ 3 ++ 2 +++ 2 +++ 2 rh43 ++ 3 ++++ 1 ++++ 2 +++ 3 + 3 ++++3 +++ 2 +++ 1 Scoring: (−) no transduction, (+) very few positive cells(++) some positive cells, (+++) many positive cells, and (++++) regionis almost saturated with EGFP-positive cells. The number of animals (n)with the particular score is given to the right of the score.

Example 10: Evaluation of an rAAV Based Treatment in a Canavan DiseaseModel Introduction to the Example

CD is a rare and fatal childhood leukodystrophy caused by autosomalrecessive mutations in the aspartoacylase gene (ASPA) [as established byG.G.'s graduate work (12)]. ASPA deficiency in CD patients leads toelevated N-Acetyl-Aspartic Acid (NAA) in urine (a hallmark of CD) andspongy degeneration of white matter throughout the CNS, producing severepsychomotor retardation and early death. An ASPA^(−/−) mouse modelmimics the neuropathology and clinical manifestations seen in CDpatients, i.e., spongy degeneration of white matter, motor deficits,developmental delays, and early death (within 3 weeks after birth).

In this study, i.v. deliverable rAAVs were used to target the CNSglobally to treat diffused WM degeneration in CD mice. Single i.v.injections of ASPA vector to the neonatal CD mice corrected metabolicdefect, psychomotor malfunction and other disease phenotypes, andprolonged survival. While untreated CD mice started showing growthretardation, psychomotor malfunction in the 2^(nd) wk after birth anduniformly died soon after weaning, the treated mice began to gain weight2 wks after vector injection and nearly caught up with theirheterozygous littermates within 7-8 weeks. Unlike CD mice, the mobilityof the treated animals was similar to Wt littermates. Data from rotarodtest on the treated mice showed no significant differences in thelatency time among the treated CD mice and their age-matched Wtlittermates, indicating that gene therapy corrected the ataxia, atypical neuromuscular symptom of CD. Biochemical characterizationindicated reduction of NAA levels in the urine samples and restorationof ASPA activity in their brain and kidney tissues. Mitigation of thebiochemical and clinical phenotypes was well correlated with globallyameliorated histopathology in not only the brain, spinal cord but alsoin the peripheral tissues such as kidney, indicating that CD is not justa CNS disorder.

Results

In CD mice were dosed at P1 (facial vein, 4×10¹¹ GCs) with AAV9ASPA. Themice were monitored for growth, gait, motor function on rotarod, NAAlevels in urine and ASPA activities in brain. The results showed that i)Untreated CD mice started losing weight at the 2^(nd) week and died inthe 3^(rd) week after birth; ii) The treated animals recovered theircapacity to grow in the 5^(th) week and caught up with ASPA^(+/−) animalby the 10^(th) week; iii). Gene therapy completely corrected gait of CDmice as well as motor function of the CD mice treated at P1 (FIG. 30A)as measured by rotarod test; iv). Gene therapy restored the vision of CDmice. The electroretinography (ERG) tests on the eyes of the CD miceshowed non-recordable responses to light, while well-defined ERGresponses were readily detectable in the treated CD mice (FIG. 30B).These data indicate a severer retinopathy and loss of vision in CD miceand gene therapy can mitigate the retinopathy and restore the vision ofCD mice; v). Gene therapy clearly improved metabolic defects of NAA asthe NAA levels in the treated CD mice approach those in the control mice(FIG. 30C); and vi) correction of NAA metabolism is well correlated withrestoration of ASPA expression (FIG. 30E) and activities (FIG. 30D) inthe brain of the treated CD mice.

To determine if the phenotypic corrections are correlated withalleviated neuropathology as well as in situ expression of ASPA in thebrain sections of the treated CD mice, brain sections were analyzed at 3months after gene therapy for neuropathology and ASPAimmunohistochemistry. While the untreated mouse brain shows markedvacuolation that diffusely involves all regions of the brain and spinalcord, the vacuolation in both brain and spinal cord of the treatedanimal appears more patchy and variable with generally smaller-sizedvacuoles. Some areas of the cerebral cortex show almost no vacuolation(FIG. 31A). In addition, avidin-biotin complex (ABC) system was used tostain brain sections to detect ASPA expression in the cerebral cortex insitu (FIG. 31B). To generate quantitative measurements on theimprovement of neuropathology in the treated CD mice, the “vacuoles” inthe brain and spinal cord sections caused by the white matterdegeneration in the CD mice were quantified before and after genetherapy treatment. For this quantitative analysis, a Nikon Eclipse Tiinverted microscope and Nikon NIS elements AR software V.3.2 were used.Vacuoles that were >3,000 pixels, 1,000-3,000 pixels and 100-1,000pixels were defined as large, medium and small vacuoles respectively.Among the 5 brain regions evaluated in this experiment, the olfactorybulb had the most dramatic mitigation in the white matter degenerationafter gene therapy (FIG. 32A). For the other 3 regions, while the largevacuoles were completely eliminated and the numbers of medium vacuoleswere remarkably reduced, the reduction in the numbers of small vacuoles(<100 um) was not as significant in this experiment (FIG. 32A). The sameanalysis on the spinal cord sections revealed a similar trend (FIG.32B).

Histopathology of the kidneys in the CD mice were evaluated. Theglomeruli showed normal structure but were associated with dilation ofBowman's spaces. The renal tubular epithelium was diffusely attenuated(or atrophic) in association with enlargement of the tubular lumens(FIG. 33A). In contrast, the treated CD mouse had normal glomeruli.Renal tubular epithelial cells were well-stained and normal in volume(FIG. 33B). These results indicate the involvement of kidney in thepathophysiology of CD and kidney as a peripheral target for CD genetherapy. This result also indicates renal tropism of AAV vectors as aconsideration for selection of a vector for CD gene therapy. Twovectors, rAAV9 and rh.10 were evaluated for efficiency of kidneytransduction after IV delivery to 10 week old C57BL/6 mice. The resultsindicate the use of rAAVrh.10 (FIG. 33D) as a useful vector for CD genetherapy because it transduces kidney efficiently in addition to itsefficient CNS transduction (FIG. 33C).

NUCLEIC ACID AND AMINO ACID SEQUENCES >gi|9632548|ref|NP_049542.1|capsid protein [Adeno-associated virus-1] (SEQ ID NO: 1)MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL >gi|110645923|ref|YP_680426.1|major coat protein VP1 [Adeno-associated virus-2] (SEQ ID NO: 2)MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL >gi|51593838|ref|YP_068409.1|capsid protein [Adeno-associated virus-5] (SEQ ID NO: 3)MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL >gi|2766607|gb|AAB95450.1|capsid protein VP1 [Adeno-associated virus-6] (SEQ ID NO: 4)MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL >gi|171850125|gb|ACB55302.1|capsid protein VP1 [Adeno-associated virus- 6.2] (SEQ ID NO: 5)MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL >gi|22652861|gb|AAN03855.1|AF513851_2 capsid protein [Adeno-associated virus-7] (SEQ ID NO: 6)MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPAKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSSVGSGTVAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSETAGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLRFKLFNIQVKEVTTNDGVTTIANNLTSTIQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTGNNFEFSYSFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSNPGGTAGNRELQFYQGGPSTMAEQAKNWLPGPCFRQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIFGKTGATNKTTLENVLMTNEEEIRPTNPVATEEYGIVSSNLQAANTAAQTQVVNNQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNFEKQTGVDFAVDSQGVYSEPRPIGTRYLTRNL >gi|22652864|gb|AAN03857.1|AF513852 2 capsid protein [Adeno-associated  virus-8] (SEQ ID NO: 7)MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL >gi|46487805|gb|AAS99264.1|capsid protein VP1 [Adeno-associated virus 9] (SEQ ID NO: 8)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL >gi|29650526|gb|AAO88201.1| capsid protein [Non-human primate Adeno-associated virus] (SEQ ID NO: 9) rh-10MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL >gi|171850147|gb|ACB55313.1|capsid protein VP1 [Adeno-as sociated virus- rh.39] (SEQ ID NO: 10)MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEAAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTQGTQQLLFSQAGPANMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGRDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQTNTGPIVGNVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL >gi|46487767|gb|AAS99245.1|capsid protein VP1 [Adeno-associated virus rh.43] (SEQ ID NO: 11)MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLEAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPVTGSCFWQQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL >capsid protein VP1 [Adeno-associated virus] CSp3 (SEQ ID NO: 12)MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTIASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKRISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIRVKEVTDNNGVKTITNNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTRNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGVKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL >gi|189339202|ref|NP_001121557.1| aspartoacylase [Homo sapiens](SEQ ID NO: 13)MTSCHIAEEHIQKVAIFGGTHGNELTGVFLVKHWLENGAEIQRTGLEVKPFITNPRAVKKCTRYIDCDLNRIFDLENLGKKMSEDLPYEVRRAQEINHLFGPKDSEDSYDIIFDLHNTTSNMGCTLILEDSRNNFLIQMFHYIKTSLAPLPCYVYLIEHPSLKYATTRSIAKYPVGIEVGPQPQGVLRADILDQMRKMIKHALDFIHHFNEGKEFPPCAIEVYKIIEKVDYPRDENGEIAAIIHPNLQDQDWKPLHPGDPMFLTLDGKTIPLGGDCTVYPVFVNEAAYYEKKEAFAKTTKLTLNAKSIRCCLH>gi|189339201:92-1033 Homo sapiens aspartoacylase (Canavan disease) (ASPA),transcript variant 2, mRNA (SEQ ID NO: 14)ATGACTTCTTGTCACATTGCTGAAGAACATATACAAAAGGTTGCTATCTTTGGAGGAACCCATGGGAATGAGCTAACCGGAGTATTTCTGGTTAAGCATTGGCTAGAGAATGGCGCTGAGATTCAGAGAACAGGGCTGGAGGTAAAACCATTTATTACTAACCCCAGAGCAGTGAAGAAGTGTACCAGATATATTGACTGTGACCTGAATCGCATTTTTGACCTTGAAAATCTTGGCAAAAAAATGTCAGAAGATTTGCCATATGAAGTGAGAAGGGCTCAAGAAATAAATCATTTATTTGGTCCAAAAGACAGTGAAGATTCCTATGACATTATTTTTGACCTTCACAACACCACCTCTAACATGGGGTGCACTCTTATTCTTGAGGATTCCAGGAATAACTTTTTAATTCAGATGTTTCATTACATTAAGACTTCTCTGGCTCCACTACCCTGCTACGTTTATCTGATTGAGCATCCTTCCCTCAAATATGCGACCACTCGTTCCATAGCCAAGTATCCTGTGGGTATAGAAGTTGGTCCTCAGCCTCAAGGGGTTCTGAGAGCTGATATCTTGGATCAAATGAGAAAAATGATTAAACATGCTCTTGATTTTATACATCATTTCAATGAAGGAAAAGAATTTCCTCCCTGCGCCATTGAGGTCTATAAAATTATAGAGAAAGTTGATTACCCCCGGGATGAAAATGGAGAAATTGCTGCTATCATCCATCCTAATCTGCAGGATCAAGACTGGAAACCACTGCATCCTGGGGATCCCATGTTTTTAACTCTTGATGGGAAGACGATCCCACTGGGCGGAGACTGTACCGTGTACCCCGTGTTTGTGAATGAGGCCGCATATTACGAAAAGAAAGAAGCTTTTGCAAAGACAACTAAACTAACGCTCAATGCAAAAAGTATTCGCTGCTGTTTACATTAG >gi|31560279|ref|NP_075602.2| aspartoacylase [Mus Musculus](SEQ ID NO: 15)MTSCVAKEPIKKIAIFGGTHGNELTGVFLVTHWLRNGTEVHRAGLDVKPFITNPRAVEKCTRYIDCDLNRVFDLENLSKEMSEDLPYEVRRAQEINHLFGPKNSDDAYDLVFDLHNTTSNMGCTLILEDSRNDFLIQMFHYIKTCMAPLPCSVYLIEHPSLKYATTRSIAKYPVGIEVGPQPHGVLRADILDQMRKMIKHALDFIQHFNEGKEFPPCSIDVYKIMEKVDYPRNESGDMAAVIHPNLQDQDWKPLHPGDPVFVSLDGKVIPLGGDCTVYPVFVNEAAYYEKKEAFAKTTKLTLSAKSIRSTLH>gi|142354273:148-1086 Mus musculus aspartoacylase (Aspa), mRNA(SEQ ID NO: 16) ATGACCTCTTGTGTTGCTAAAGAACCTATTAAGAAGATTGCCATCTTTGGAGGGACTCATGGAAATGAACTGACCGGAGTGTTTCTAGTTACTCACTGGCTAAGGAATGGCACTGAAGTTCACAGAGCAGGGCTGGACGTGAAGCCATTCATTACCAATCCAAGGGCGGTGGAGAAGTGCACCAGATACATTGACTGTGACCTGAATCGTGTTTTTGACCTTGAAAATCTTAGCAAAGAGATGTCTGAAGACTTGCCATATGAAGTGAGAAGGGCTCAAGAAATAAATCATTTATTTGGTCCAAAAAATAGTGATGATGCCTATGACCTTGTTTTTGACCTTCACAACACCACTTCTAACATGGGTTGCACTCTTATTCTTGAGGATTCCAGGAATGACTTTTTAATTCAGATGTTTCACTATATTAAGACTTGCATGGCTCCATTACCCTGCTCTGTTTATCTCATTGAGCATCCTTCACTCAAATATGCAACCACTCGTTCCATTGCCAAGTATCCTGTTGGTATAGAAGTTGGTCCTCAGCCTCACGGTGTCCTTAGAGCTGATATTTTAGACCAAATGAGAAAAATGATAAAACATGCTCTTGATTTTATACAGCATTTCAATGAAGGAAAAGAATTTCCTCCCTGTTCTATTGACGTCTATAAAATAATGGAGAAAGTTGATTATCCAAGGAATGAAAGTGGAGACATGGCTGCTGTTATTCATCCTAATCTGCAGGATCAAGACTGGAAACCATTGCACCCTGGAGATCCTGTGTTTGTGTCTCTTGATGGAAAAGTTATTCCACTGGGTGGAGACTGTACCGTGTACCCAGTGTTTGTGAATGAAGCTGCATATTATGAAAAAAAAGAAGCATTTGCAAAGACAACAAAACTAACACTCAGCGCAAAAAGCATCCGCTCCACTTTGCAC TAA>gi|48762945:149-613 Homo sapiens superoxide dismutase 1, soluble (SOD1),mRNA (SEQ ID NO: 17)ATGGCGACGAAGGCCGTGTGCGTGCTGAAGGGCGACGGCCCAGTGCAGGGCATCATCAATTTCGAGCAGAAGGAAAGTAATGGACCAGTGAAGGTGTGGGGAAGCATTAAAGGACTGACTGAAGGCCTGCATGGATTCCATGTTCATGAGTTTGGAGATAATACAGCAGGCTGTACCAGTGCAGGTCCTCACTTTAATCCTCTATCCAGAAAACACGGTGGGCCAAAGGATGAAGAGAGGCATGTTGGAGACTTGGGCAATGTGACTGCTGACAAAGATGGTGTGGCCGATGTGTCTATTGAAGATTCTGTGATCTCACTCTCAGGAGACCATTGCATCATTGGCCGCACACTGGTGGTCCATGAAAAAGCAGATGACTTGGGCAAAGGTGGAAATGAAGAAAGTACAAAGACAGGAAACGCTGGAAGTCGTTTGGCTTGTGGTGTAATTGGGATCGCCCAATAA >gi|4507149|ref|NP_000445.1|superoxide dismutase [Homo sapiens] (SEQ ID NO: 18)MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ>gi|45597446:117-581 Mus musculus superoxide dismutase 1, soluble (Sod1),mRNA (SEQ ID NO: 19)ATGGCGATGAAAGCGGTGTGCGTGCTGAAGGGCGACGGTCCGGTGCAGGGAACCATCCACTTCGAGCAGAAGGCAAGCGGTGAACCAGTTGTGTTGTCAGGACAAATTACAGGATTAACTGAAGGCCAGCATGGGTTCCACGTCCATCAGTATGGGGACAATACACAAGGCTGTACCAGTGCAGGACCTCATTTTAATCCTCACTCTAAGAAACATGGTGGCCCGGCGGATGAAGAGAGGCATGTTGGAGACCTGGGCAATGTGACTGCTGGAAAGGACGGTGTGGCCAATGTGTCCATTGAAGATCGTGTGATCTCACTCTCAGGAGAGCATTCCATCATTGGCCGTACAATGGTGGTCCATGAGAAACAAGATGACTTGGGCAAAGGTGGAAATGAAGAAAGTACAAAGACTGGAAATGCTGGGAGCCGCTTGGCCTGTGGAGTGATTGGGATTGCGCAGTAA >gi|45597447|ref|NP_035564.1|superoxide dismutase [Mus musculus] (SEQ ID NO: 20)MAMKAVCVLKGDGPVQGTIHFEQKASGEPVVLSGQITGLTEGQHGFHVHQYGDNTQGCTSAGPHFNPHSKKHGGPADEERHVGDLGNVTAGKDGVANVSIEDRVISLSGEHSIIGRTMVVHEKQDDLGKGGNEESTKTGNAGSRLACGVIGIAQ >pAAVscCB6 EGFPmir SOD5 (direct) 5243 bp (SEQ ID NO: 21)CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCATGCTCTAGGAAGATCAATTCAATTCACGCGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGATATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTCTAGCCTCGAGAATTCACGCGTGGTACCTCTAGAGCAGAGCTCGTTTAGTGAACCGTCAGTTCGAAATCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGTAACAGGTAAGTGCGATCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTGTCGTCTGTTGCGGGGGCGGCAGTTATGGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGTACCTGCTGTTGACAGTGAGCGACGCAATGTGACTTCGCTGACAAAGCTGTGAAGCCACAGATGGGCTTTGTCAGCAGTCACATTGCGCTGCCTACTGCCTCGGACTTCAAGGGCTCGAGAATTCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGCGGCCGCAACAACGCGTTCCTGACCATTCATCCTCTTTCTTTTTCCTGCAGGCTTGTGGAAGAAATGGGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGCCTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG >sod1mir1 (direct) 108 bp(SEQ ID NO: 22) TGCTGTTGACAGTGAGCGACATCATCAATTTTCCGAGCAGAACTGTGAAGCCACAGATGGGTTCTGCTCGAAATTGATGATGCTGCCTACTGCCTCGGACTTCAAGGG >sod1mir2 (direct) 106 bp(SEQ ID NO: 23) TGCTGTTGACAGTGAGCGACGCATTAAAGGATCCTGACTGACTGTGAAGCCACAGATGGGTCAGTCAGTCCTTTAATGCGCTGCCTACTGCCTCGGACTTCAAGGG >sod1mir3 (direct) 108 bp(SEQ ID NO: 24) TGCTGTTGACAGTGAGCGACTGCATGGATTCTCCATGTTCATCTGTGAAGCCACAGATGGGATGAACATGGAATCCATGCAGCTGCCTACTGCCTCGGACTTCAAGGG >sod1mir4 (direct) 106 bp(SEQ ID NO: 25) TGCTGTTGACAGTGAGCGACAAGGATGAAGATCGAGGCATGCTGTGAAGCCACAGATGGGCATGCCTCTCTTCATCCTTGCTGCCTACTGCCTCGGACTTCAAGGG >sod1mir5 (direct) 110 bp(SEQ ID NO: 26) TGCTGTTGACAGTGAGCGACGCAATGTGACTTCGCTGACAAAGCTGTGAAGCCACAGATGGGCTTTGTCAGCAGTCACATTGCGCTGCCTACTGCCTCGGACTTCAAGGG >sod1mir6 (direct) 108 bp (SEQ ID NO: 27)TGCTGTTGACAGTGAGCGACCGATGTGTCTATCTTGAAGATTCTGTGAAGCCACAGATGGGAATCTTCAATAGACACATCGGCTGCCTACTGCCTCGGACTTCAAGGG >sod1mir7 (direct) 106 bp(SEQ ID NO: 28) TGCTGTTGACAGTGAGCGACGGTGGAAATGATCAGAAAGTACTGTGAAGCCACAGATGGGTACTTTCTTCATTTCCACCGCTGCCTACTGCCTCGGACTTCAAGGG >sod1mir8 (direct) 110 bp(SEQ ID NO: 29) TGCTGTTGACAGTGAGCGACGCTGTAGAAATTCGTATCCTGATCTGTGAAGCCACAGATGGGATCAGGATACATTTCTACAGCGCTGCCTACTGCCTCGGACTTCAAGGG >sod1mir9 (direct) 106 bp(SEQ ID NO: 30) TGCTGTTGACAGTGAGCGAGGTATTAAACTTGTCAGAATTTAGTGAAGCCACAGATGTAAATTCTGACAAGTTTAATACCCTGCCTACTGCCTCGGACTTCAAGGG >pAAVscCB6 EGFPmir scr (1820 bp-1925 bp, direct) 106 bp(SEQ ID NO: 31) TGCTGTTGACAGTGAGCGACGATGCTCTAATCGGTTCTATCAAGTGAAGCCACAGATGTTGATAGAACCTTAGAGCATCGCTGCCTACTGCCTCGGACTTCAAGGG

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thisdescription or illustrated in the drawings. The invention is capable ofother embodiments and of being practiced or of being carried out invarious ways. Also, the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having,” “containing,”“involving,” and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1-55. (canceled)
 56. A method for reducing the severity or extent of deficiency of aspartoacylase in a subject, the method comprising: intrathecally, intracerebrally, or intravascularly administering rAAV to the subject, wherein the rAAV comprises a nucleic acid comprising a promoter operably linked with a region encoding aspartoacylase (ASPA), wherein the ASPA is expressed from the nucleic acid in cells transduced by the rAAV.
 57. The method of claim 56, wherein the nucleic acid expresses an aspartoacylase (ASPA) mRNA comprising one or more miRNA binding sites for one or more miRNAs that are more abundant in one or more non-CNS tissues in comparison to a CNS tissue.
 58. The method of claim 57, wherein the one or more miRNAs that are more abundant in one or more non-CNS tissues in comparison to the CNS tissue are at least twofold more abundant.
 59. The method of claim 56, wherein the rAAV is administered intrathecally to the subject.
 60. The method of claim 56, wherein the rAAV is administered intracerebrally to the subject.
 61. The method of claim 56, wherein the rAAV is administered intravascularly to the subject.
 62. The method of claim 56, wherein the rAAV comprises a capsid of either SEQ ID NO: 7 or
 8. 63. The method of claim 56, wherein the rAAV is self-complementary.
 64. The method of claim 56, wherein the rAAV is delivered to an area of the subject selected from the group consisting of brain tissue, meninges, neuronal cells, glial cells, astrocytes, oligodendrocytes, microglial cells, ependymal cells, Schwann cells, cerebrospinal fluid (CSF), and interstitial spaces.
 65. The method of claim 56, wherein oligodendrocytes, astrocytes, microglial cells, ependymal cells, Schwann cells, glial cells, neuronal cells or other central nervous system cells are transduced.
 66. The method of claim 56, wherein the dose of rAAV for intrathecal administration is in the range of 10¹⁰ genome copies per kilogram to 10¹⁴ genome copies per kilogram.
 67. The method of claim 56, wherein the dose of rAAV for intracerebral administration is in the range of 10¹⁰ genome copies per kilogram to 10¹⁴ genome copies per kilogram.
 68. The method of claim 56, wherein the dose of rAAV for intravascular administration is in the range of 10¹⁰ genome copies per kilogram to 10¹⁴ genome copies per kilogram.
 69. The method of claim 56, wherein the promoter is inducible.
 70. The method of claim 56, wherein the promoter is constitutive.
 71. The method of claim 56, wherein the promoter is tissue-specific.
 72. The method of claim 56, wherein administration is by both intravascularly and intracerebrally.
 73. A method for reducing the severity or extent of deficiency of aspartoacylase in a subject, the method comprising: intrathecally, intracerebrally, or intravascularly administering rAAV to the subject an rAAV comprises (i) a capsid protein having the amino acid sequence of SEQ ID NO: 8 and (ii) a nucleic acid comprising a constitutive promoter operably linked with a region encoding aspartoacylase (ASPA), wherein the ASPA is expressed from the nucleic acid in cells transduced by the rAAV.
 74. The method of claim 73, wherein the nucleic acid expresses an aspartoacylase (ASPA) mRNA comprising one or more miRNA binding sites for one or more miRNAs that are more abundant in one or more non-CNS tissues in comparison to a CNS tissue.
 75. The method of claim 74, wherein the one or more miRNAs that are more abundant in one or more non-CNS tissues in comparison to the CNS tissue are at least twofold more abundant.
 76. The method of claim 73, wherein the rAAV is administered intrathecally to the subject.
 77. The method of claim 73, wherein the rAAV is administered intracerebrally to the subject.
 78. The method of claim 73, wherein the rAAV is administered intravascularly to the subject.
 79. The method of claim 73, wherein the rAAV is self-complementary.
 80. The method of claim 73, wherein the rAAV is delivered to an area of the subject selected from the group consisting of brain tissue, meninges, neuronal cells, glial cells, astrocytes, oligodendrocytes, microglial cells, ependymal cells, Schwann cells, cereobrospinal fluid (CSF), and interstitial spaces.
 81. The method of claim 73, wherein oligodendrocytes, astrocytes, microglial cells, ependymal cells, Schwann cells, glial cells, neuronal cells or other central nervous system cells are transduced.
 82. The method of claim 73, wherein the dose of rAAV for intrathecal administration is in the range of 10¹⁰ genome copies per kilogram to 10¹⁴ genome copies per kilogram.
 83. The method of claim 73, wherein the dose of rAAV for intracerebral administration is in the range of 10¹⁰ genome copies per kilogram to 10¹⁴ genome copies per kilogram.
 84. The method of claim 73, wherein the dose of rAAV for intravascular administration is in the range of 10¹⁰ genome copies per kilogram to 10¹⁴ genome copies per kilogram.
 85. The method of claim 73, wherein administration is by both intravascularly and intracerebrally. 